Device and method of restoring microbiota of newborns

ABSTRACT

The invention relates to methods and compositions for restoring normal microbiota in pre-term newborns or newborns delivered by Cesarean section and methods for preventing or ameliorating diseases associated with delivery by Cesarean section or pre-term birth comprising administering to said newborns at the time of birth or shortly thereafter an effective amount of a vaginal microbiota inoculum obtained from the newborn&#39;s mother or a donor or an effective amount of a probiotic composition. The invention also relates to a device for collecting maternal vaginal microbiota from the vaginal canal of a patient. The device can have a housing forming a cavity, an absorbent material removable disposed within the cavity, and a deployment element disposed in the housing and movable along a length of the housing. The movement of the deployment element can displace the absorbent material out of the cavity and in to the vaginal canal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of International PatentApplication No. PCT/US16/68735, filed Dec. 27, 2016, which published asWO 2017/117142 A1 on Jul. 6, 2017, which claims priority to U.S.Provisional Application No. 62/271,692, filed on Dec. 28, 2015, thedisclosure of which are herein incorporated by reference in theirentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant DK090989awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 22, 2016, isnamed 243735.000163_SL.txt and is 5,816 bytes in size.

FIELD OF THE INVENTION

The application relates to methods and devices for restoring normalmicrobiota in pre-term infants or infants delivered by Cesarean sectionand methods for preventing or ameliorating diseases associated withdelivery by Cesarean section or pre-term birth comprising administeringto said infants at the time of birth or shortly thereafter an effectiveamount of a vaginal microbiota inoculum obtained from the infant'smother or a donor during the third trimester of pregnancy before or atthe time of giving birth or an effective amount of a probioticcomposition, wherein said probiotic composition (i) stimulates growthand/or activity of bacteria which are under-represented in microbiota ofsaid newborn as compared to vaginally delivered full-term newborns,and/or (ii) inhibits growth and/or activity of bacteria which areover-represented in microbiota of said newborn as compared to vaginallydelivered full-term newborns. Also provided are methods for diagnosingabnormal microbiota development in a newborn, comprising determining arelative abundance of one or more bacterial taxa in a microbiota sampleobtained from said newborn. Also provided are methods of using devicesto assist in restoring normal natural maternal vaginal microbiota innewborns under non-natural vaginal delivery, and/or in pre-term orpremature infants or infants delivered by Cesarean section.

BACKGROUND

Maternal vaginal microbes provide the natural seeding to the newbornmicrobiota (1). Whether vaginal microbes can reach the placenta and thefetus before labor initiates, still unclear (2, 3), but mode of deliveryoverwhelms any other possible previous signal, and C-section-born babiesare microbiologically different from vaginally born infants (1). Thematernal vaginal (4) and intestinal (5) microbiota change during thethird trimester of pregnancy, but the significance of these changes forthe fitness of the baby has not been understood. Early interaction withindigenous microbes is essential for healthy immunological and metabolicprogramming, and contact with bacterial populations in the vagina duringbirth marks the beginning of eventual massive bacterial colonization ofthe newborn's mucosal surfaces. Mucosal immunity is strongly influencedby the microbiota (6), which in the gut mucosa, is subject to continuoussurveillance by M cells—from the Peyer's patches of the gut-associatedlymphoid tissue (GALT)—for processing by local dendritic cells andsubsequently modulate CD4+ to produce Tregs and induce tolerance.

The “education” of the immune system by the microbiota starts at thevery first microbial exposure and pioneer bacteria probably play adeterminant role. Few studies have focused on the development of theintestinal microbiota and immunity (7-10), but the assembly in multiplebody sites, the fate of vaginal lactic acid bacterial populations, andthe ecological dynamics between maternal and infant communities thatinteract are not known. Despite the vast descriptive knowledge about themicrobiota of American adults generated by the HMP project (NIH), littleis known about developmental aspects of the microbiota.

The incidence of immune, inflammatory, and metabolic disorders isincreasing in industrialized countries (11, 12). Early life events andaberrant microbial colonization has been associated with these diseases(12, 13). Epidemiological associations show C-section delivery increasedrisk of asthma and allergies (14-21), intestinal inflammatory conditions(19, 22-24). Obesity and diabetes (type I) is also increased byC-section birthing (19, 25-29) and by antibiotic consumption (19, 27-30)even as early as during gestation (25).

The phenomenon of decreased oral and gut microbial diversity in thefirst days after birth, previously reported in mice (36), is of unknownfunctional significance, but might be reflecting the selective effect ofmilk on the gut microbiota. Bacteroides, Clostridiales andBifidobacterium are bacteria that are enriched in the gut during thefirst weeks of life, during strict lactation.

Cesarean section birthing without maternal membrane rupture, as in thecase of scheduled Cesarean, impedes the seeding of the babies withvaginal microbes. C-section is medically indicated in 13-15% of thebirths, saving many lives of mothers and babies. However, scheduledC-section is becoming the standard of birthing in many countries of theworld, with over 50% of births in Brazil, Dominican Republic, and Iranand many other countries approaching these rates (Health at a Glance2011: OECD Indicators; WHO Global Health Observatory; (31); (37)).C-section birthing is associated with short health risks for the motherand baby and with long term health risks for the babies, includingceliac disease (23, 24), asthma (16, 20, 21, 38), type 1 diabetes (39,40), and obesity (32, 41, 42).

Thus, there is a great need in the art for a device and method tocapture and deliver, restoring, and/or improving normal microbiota froma mother to their newborn, particularly those under non-naturallyvaginal delivery, and/or born in pre-term or premature.

SUMMARY OF THE INVENTION

As specified in the Background section above, there is a great need inthe art for preventing or ameliorating diseases associated with deliveryby Cesarean section or pre-term birth. The present application addressesthese and other needs by providing methods for restoring normalmicrobiota in pre-term newborns or newborns delivered by Cesareansection and methods for treating (e.g., preventing or ameliorating)diseases associated with delivery by Cesarean section or pre-term birthcomprising administering to said newborns at the time of birth orshortly thereafter an effective amount of maternal vaginal microbiotainoculum or an effective amount of a probiotic composition, wherein saidprobiotic composition (i) stimulates growth and/or activity of bacteriawhich are under-represented in microbiota of said newborn as compared tovaginally delivered full-term newborns, and/or (ii) inhibits growthand/or activity of bacteria which are over-represented in microbiota ofsaid newborn as compared to vaginally delivered full-term newborns.

In one aspect, the invention provides a method for restoring normalmicrobiota in an infant delivered by Cesarean section, said methodcomprising administering to said infant at the time of birth and/orwithin the first 4 months of life (preferably, within the first 24 hoursof life, most preferably within the first hour of life) an effectiveamount of a vaginal microbiota inoculum, wherein said inoculum isobtained from the subject's mother or from a donor during the thirdtrimester of pregnancy before or at the time of giving birth.

In a related aspect, the invention provides a method for restoringnormal microbiota in a pre-term infant, said method comprisingadministering to said infant at the time of birth and/or within thefirst 4 months of life (preferably, within the first 24 hours of life,most preferably, within the first hour of life) an effective amount of avaginal microbiota inoculum, wherein said inoculum is obtained from thesubject's mother or from a donor during the third trimester of pregnancybefore or at the time of giving birth.

In a further aspect, the invention provides a method for treating (e.g.,preventing or ameliorating) a disease in a subject associated with thesubject's delivery by Cesarean section or with the subject's pre-termbirth, said method comprising administering to said subject at the timeof birth and/or within the first 4 months of life an effective amount ofa vaginal microbiota inoculum, wherein said inoculum is obtained fromthe subject's mother or from a donor during the third trimester ofpregnancy before or at the time of giving birth. In one embodiment, saiddisease is an inflammatory or an autoimmune disorder. In one embodiment,said disease is selected from the group consisting of autoimmunediseases, allergic diseases, infectious diseases, and rejection in organtransplantations. In one specific embodiment, said disease is selectedfrom the group consisting of asthma, allergy, celiac disease, type 1diabetes, obesity, necrotizing enterocolitis, inflammatory bowel disease(IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis,rheumatoid arthritis, multiple sclerosis, graft vs. host diseasefollowing bone marrow transplantation, osteoarthritis, juvenile chronicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, insulin dependentdiabetes mellitus, thyroiditis, asthma, psoriasis, dermatitisscleroderma, atopic dermatitis, graft versus host disease, acute orchronic immune disease associated with organ transplantation,sarcoidosis, and atherosclerosis.

In one embodiment of any of the above methods of the invention, thevaginal microbiota inoculum is delivered to the mouth, nose, or skin ofthe infant. In one embodiment of any of the above methods of theinvention, the vaginal microbiota is inoculum administered to the infantby a route selected from the group consisting of oral, topical, rectal,mucosal, sublingual, nasal, and via naso/oro-gastric gavage. In oneembodiment of any of the above methods of the invention, the vaginalmicrobiota is administered to the infant by placing it on the maternalbreast and/or chest.

In one embodiment of any of the above methods of the invention, thevaginal microbiota inoculum is delivered to the infant in a form of aliquid, foam, cream, spray, powder, or gel. In one embodiment of any ofthe above methods of the invention, the vaginal microbiota inoculum isdelivered to the infant in a form of a composition which comprises (i) acarrier and/or excipient and/or (ii) one or more prebiotic agents whichstimulate growth and/or activity of one or more bacteria present in thecomposition. In one specific embodiment, said composition comprises abuffering agent to adjust pH to the natural vaginal pH at the time oflabor or to a pH of 3.5 to 7. In one specific embodiment, saidcomposition comprises an excipient or a carrier that optimizes theseeding of the transferred microbiota.

In one embodiment of any of the above methods of the invention, thevaginal microbiota inoculum is delivered using an absorbent material ordevice (e.g., gauze, sponge, or tampon). In one specific embodiment, thevaginal microbiota inoculum is transferred to said absorbent material ordevice by introducing said absorbent material or device (e.g., for atleast 5 minutes) in vagina prior to the birth or at the time of Cesareansection.

In one embodiment of any of the above methods, the vaginal microbiotainoculum, after it is obtained from the subject's mother or the donor,is stored in a frozen form.

In one embodiment of any of the above methods, the vaginal microbiotainoculum, after it is obtained from the subject's mother or the donor,is processed to isolate desired bacteria as single or mixed cultures andsuch mixed or single cultures are then administered to the infant.

In one embodiment of any of the above methods, the vaginal microbiotainoculum is lyophilized after it is obtained from the subject's motheror the donor and reconstituted prior to the administration to theinfant.

In one embodiment of any of the above methods, prior to obtainingvaginal microbiota from the newborn's mother or the donor, it isverified that said mother or donor does not have Group B Streptococcus(GBS) and/or sexually transmitted diseases such as, e.g., humanimmunodeficiency virus (HIV) and/or Chlamydia. In one embodiment of anyof the above methods, prior to obtaining vaginal microbiota from thenewborn's mother or the donor, it is verified that said mother's ordonor's vaginal pH is less than 4.5.

In one embodiment of any of the above methods of the invention, thenewborn's mother or the donor has not been administered antibioticcompounds within a certain period prior to the collection of themicrobiota (preferably, for at least one month prior to the collectionof the microbiota), is not obese or overweight (preferably has body massindex (BMI) scores of below 25, most preferably between 18.5 and 24.9),and does not have irritable bowel disease, Crohn's disease, ulcerativecolitis, irritable bowel syndrome, celiac disease, colorectal cancer,and a family history of these diseases.

In one embodiment of any of the above methods, the method furthercomprises monitoring the infant's microbiota after the administration ofthe vaginal microbiota by: (a) determining a relative abundance of oneor more bacterial taxa in a microbiota sample obtained from the infant(e.g., isolated from feces, skin, oral mucosa, conjunctive mucosa, ornasal mucosa), and (b) comparing the relative abundance(s) determined instep (a) to (i) a predetermined standard value or (ii) to theabundance(s) of the same taxa in a control subject (e.g., a vaginallydelivered full-term healthy infant) or (iii) to the average value ofabundances of the same taxa in several control subjects. Non-limitingexamples of the methods which can be used for determining the relativeabundance of the bacterial taxa include, e.g., quantitative polymerasechain reaction (qPCR), sequencing of bacterial 16S rRNA, shotgunmetagenome sequencing, and metabolomics. In one specific embodiment, themethod involves determining a relative abundance of one or more bacteriafrom the taxa selected from the group consisting of Lactobacillus,Bacteriodales, Bacteroides, Parabacteroides, Bacteroidacea,Porphyromonadaceae, Coriobacteriales, Bifidobacterium, Clostridiaceae,Stenotrophomonas, and Gemella. In one specific embodiment, the methodinvolves determining a relative abundance of one or more bacteria fromthe taxa recited in Table 1A. In one specific embodiment, the methodinvolves determining a relative abundance of bacterial species recitedin Table 1B. In one specific embodiment, the method involves determininga relative abundance of one or more bacteria from the familyNeisseriaceae.

In a separate aspect, the invention provides a method for restoringnormal microbiota in an infant delivered by Cesarean section, saidmethod comprising administering to said infant at the time of birthand/or within the first 4 months of life an effective amount of aprobiotic composition, wherein said probiotic composition (i) stimulatesgrowth and/or activity of bacteria which are under-represented inmicrobiota of said infant as compared to vaginally delivered full-terminfants, and/or (ii) inhibits growth and/or activity of bacteria whichare over-represented in microbiota of said infant as compared tovaginally delivered full-term infants.

In a related aspect, the invention provides a method for restoringnormal microbiota in a pre-term infant, said method comprisingadministering to said infant at the time of birth and/or within thefirst 4 months of life an effective amount of a probiotic composition,wherein said probiotic composition (i) stimulates growth and/or activityof bacteria which are under-represented in microbiota of said infant ascompared to vaginally delivered full-term infants, and/or (ii) inhibitsgrowth and/or activity of bacteria which are over-represented inmicrobiota of said infant as compared to vaginally delivered full-terminfants.

In another aspect, the invention provides a method for treating (e.g.,preventing or ameliorating) a disease in a subject associated with thesubject's delivery by Cesarean section or with the subject's pre-termbirth, said method comprising administering to said subject at the timeof birth and/or within the first 4 months of life a therapeuticallyeffective amount of a probiotic composition, wherein said probioticcomposition (i) stimulates growth and/or activity of bacteria which areunder-represented in microbiota of said infant as compared to vaginallydelivered full-term infants, and/or (ii) inhibits growth and/or activityof bacteria which are over-represented in microbiota of said infant ascompared to vaginally delivered full-term infants. In one embodiment,said disease is an inflammatory or an autoimmune disorder. In oneembodiment, said disease is selected from the group consisting ofautoimmune diseases, allergic diseases, infectious diseases, andrejection in organ transplantations. In one embodiment, said disease isselected from the group consisting of asthma, allergy, celiac disease,type 1 diabetes, obesity, necrotizing enterocolitis, inflammatory boweldisease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmunearthritis, rheumatoid arthritis, multiple sclerosis, graft vs. hostdisease following bone marrow transplantation, osteoarthritis, juvenilechronic arthritis, Lyme arthritis, psoriatic arthritis, reactivearthritis, spondyloarthropathy, systemic lupus erythematosus, insulindependent diabetes mellitus, thyroiditis, asthma, psoriasis, dermatitisscleroderma, atopic dermatitis, graft versus host disease, acute orchronic immune disease associated with organ transplantation,sarcoidosis, and atherosclerosis.

In one embodiment of any of the above methods involving administrationof a probiotic composition, said probiotic composition comprises one ormore bacterial strains from one or more taxa selected from the groupconsisting of Lactobacillus, Bacteriodales, Bacteroides,Parabacteroides, Bacteroidacea, Porphyromonadaceae, CoriobacterialesBifidobacterium, Clostridiaceae, Stenotrophomonas, and Gemella. In oneembodiment of any of the above methods involving administration of aprobiotic composition, said probiotic composition comprises one or morebacterial strains from one or more taxa recited in Table 1A. In oneembodiment of any of the above methods involving administration of aprobiotic composition, said probiotic composition comprises one or morebacterial strains from one or more species recited in Table 1B. In oneembodiment of any of the above methods involving administration of aprobiotic composition, said probiotic composition comprises one or moreOTUs which are independently characterized by, i.e., at least 95%, 96%,97%, 98%, 99% or including 100% sequence identity to sequences listed inSEQ ID NOS 1-12 or 16S rRNA sequences of the bacterial species recitedin Table 4 or Table 1B. In another embodiment, the OTUs may becharacterized by one or more of the variable regions of the 16S rRNAsequence (V1-V9). These regions in bacteria are defined by nucleotides69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173,1243-1294 and 1435-1465 respectively using numbering based on the E.coli system of nomenclature. (See, e.g., Brosius et al., Completenucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli,PNAS 75(10):4801-4805 (1978)). In some embodiments, at least one of theV1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterizean OTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU. In one embodiment of any of the abovemethods involving administration of a probiotic composition, saidprobiotic composition comprises one or more bacterial strains which areindependently characterized by, i.e., at least 95%, 96%, 97%, 98%, or99% sequence identity to 16S rRNA sequences of the bacterial speciesrecited in Table 1B or Table 4. In one embodiment of any of the abovemethods involving administration of a probiotic composition, saidprobiotic composition comprises one or more bacterial strains from thefamily Neisseriaceae. In one embodiment of any of the above methodsinvolving administration of a probiotic composition, said probioticcomposition augments the growth of at least one type of bacteria notdetected in the probiotic or in the newborn's gastrointestinal (GI)tract, skin, mouth or any body site, prior to administration. In oneembodiment of any of the above methods involving administration of aprobiotic composition, said probiotic composition comprises one or morebacterial strains which can be found in a healthy vaginal microbiotafrom a pregnant woman in the third trimester of pregnancy before or atthe time of giving birth. In one specific embodiment, said pregnantwoman has not been administered antibiotic compounds within a certainperiod prior to the collection of the microbiota (preferably, for atleast one month prior to the collection of the microbiota), is not obeseor overweight (preferably has body mass index (BMI) scores of below 25,most preferably between 18.5 and 24.9), and does not have irritablebowel disease, Crohn's disease, ulcerative colitis, irritable bowelsyndrome, celiac disease, colorectal cancer, and a family history ofthese diseases.

The probiotic composition useful in any of the above methods cancomprise, without limitation, e.g., live bacterial cells, conditionallylethal bacterial cells, inactivated bacterial cells, killed bacterialcells, spores (e.g., germination-competent spores), recombinant carrierstrains, cell extract, and bacterially-derived products (natural orsynthetic bacterially-derived products such as, e.g., bacterial antigensor bacterial metabolic products).

In one embodiment of any of the above methods involving administrationof a probiotic composition, said probiotic composition comprises (i) acarrier and/or excipient and/or (ii) one or more prebiotic agents whichstimulate growth and/or activity of one or more bacteria present in thecomposition. In one specific embodiment, the probiotic compositioncomprises an excipient or a carrier that optimizes the seeding of one ormore bacterial strains contained in said probiotic composition.

In one embodiment of any of the above methods involving administrationof a probiotic composition, said probiotic composition is reconstitutedfrom a lyophilized preparation. In one embodiment of any of the abovemethods involving administration of a probiotic composition, saidprobiotic composition comprises a buffering agent to adjust pH to thenatural vaginal pH at the time of labor or to a pH of 3.5 to 7.

In one embodiment of any of the above methods involving administrationof a probiotic composition, the probiotic composition is delivered tothe mouth, nose, and/or skin of the infant and/or by placing it on thematernal breast and/or chest. In one embodiment, the probioticcomposition is administered to the infant by a route selected from thegroup consisting of oral, topical, rectal (e.g., by enema), mucosal,sublingual, nasal, and via naso/oro-gastric gavage. In one embodiment,the probiotic composition is delivered to the infant in a form of aliquid, foam, cream, spray, powder, or gel. In one embodiment, theprobiotic composition is delivered using an absorbent material or device(e.g., gauze, sponge, tampon, or other applicators). In one embodiment,the probiotic composition comprises a buffering agent (e.g., sodiumbicarbonate, infant formula or sterilized human milk).

In one embodiment of any of the above methods involving administrationof a probiotic composition, the probiotic composition is administeredconjointly with a prebiotic which stimulates growth and/or activity ofbacteria contained in the probiotic composition. Non-limiting examplesof useful prebiotics include, e.g., fructooligosaccharides (FOS),galactooligosaccharides (GOS), human milk oligosaccharides (HMO),Lacto-N-neotetraose, D-Tagatose, xylo-oligosaccharides (XOS),arabinoxylan-oligosaccharides (AXOS), N-acetylglucosamine,N-acetylgalactosamine, glucose, arabinose, maltose, lactose, sucrose,cellobiose, amino acids, alcohols, resistant starch (RS), and anymixtures thereof. In one specific embodiment, the prebiotic is derivedfrom microorganisms that show stimulation by human milk components. Inone specific embodiment, the probiotic and prebiotic are administered inone composition, or simultaneously as two separate compositions, orsequentially.

In a separate embodiment, the invention provides a method for diagnosingabnormal microbiota development in an infant, comprising: (a)determining a relative abundance of one or more bacterial taxa in amicrobiota sample obtained from the infant, and (b) comparing therelative abundance(s) determined in step (a) to (i) a predeterminedstandard value or (ii) to the abundance(s) of the same taxa in a controlsubject or (iii) to the median value of abundances of the same taxa inseveral control subjects, wherein the control subject is a vaginallydelivered full-term healthy infant. Non-limiting examples of the methodswhich can be used for determining the relative abundance of thebacterial taxa include, e.g., quantitative polymerase chain reaction(qPCR), sequencing of bacterial 16S rRNA, shotgun metagenome sequencing,and metabolomics. In one embodiment, the diagnostic method involvesdetermining a relative abundance of one or more bacteria from one ormore taxa selected from the group consisting of Lactobacillus,Bacteriodales (e.g., S24-7), Bacteroides, Parabacteroides,Bacteroidacea, Porphyromonadaceae, Coriobacteriales Bifidobacterium,Clostridiaceae, Stenotrophomonas, and Gemella. In one embodiment, thediagnostic method involves determining a relative abundance of one ormore bacteria from one or more taxa present in a healthy vaginalmicrobiota from a pregnant woman in the third trimester of pregnancybefore or at the time of giving birth. In one embodiment, the diagnosticmethod involves determining a relative abundance of one or more bacteriafrom one or more taxa recited in Table 1A. In one embodiment, thediagnostic method involves determining a relative abundance of one ormore bacteria from one or more species recited in Table 1B. In oneembodiment, the diagnostic method involves determining a relativeabundance of one or more bacteria from the family Neisseriaceae.

In one embodiment of any of the above methods, the infant (or subject)is human. In one embodiment of any of the above methods, the infant is anewborn.

In another aspect, the invention provides a composition comprising (i) avaginal microbiota inoculum and (ii) a carrier and/or excipient and/orone or more prebiotic agents which stimulate growth and/or activity ofone or more bacteria present in the inoculum.

In a further aspect, the invention provides a probiotic compositioncomprising (a) one or more bacterial strains and (b) a carrier and/orexcipient and/or one or more prebiotic agents which stimulate growthand/or activity of one or more of said bacterial strains, wherein saidprobiotic composition (i) stimulates growth and/or activity of bacteriawhich are under-represented in microbiota of an infant delivered byCesarean section or born prematurely as compared to vaginally deliveredfull-term healthy infants, and/or (ii) inhibits growth and/or activityof bacteria which are over-represented in microbiota of said infant ascompared to vaginally delivered full-term healthy infants. In oneembodiment, the composition comprises two or more different bacterialstrains.

Non-limiting examples of bacteria which can be present in any of thecompositions of the invention comprise

(i) bacteria from one or more taxa selected from the group consisting ofLactobacillus, Bacteriodales (e.g., S24-7), Bacteroides,Parabacteroides, Bacteroidacea, Porphyromonadaceae, Coriobacteriales,Bifidobacterium, Clostridiaceae, Stenotrophomonas, and Gemella;(ii) bacteria from one or more taxa recited in Table 1A;(iii) bacteria from one or more species recited in Table 1B;(iv) bacteria from family Neisseriaceae.

In one embodiment of any of the compositions of the invention, thecomposition comprises one or more OTUs which are independentlycharacterized by, i.e., at least 95%, 96%, 97%, 98%, 99% or including100% sequence identity to sequences listed in SEQ ID NOS 1-12 or 16SrRNA sequences of the bacterial species recited in Table 4 or Table 1B.In another embodiment, the OTUs may be characterized by one or more ofthe variable regions of the 16S rRNA sequence (V1-V9). These regions inbacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682,822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. (See, e.g.,Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA genefrom Escherichia coli, PNAS 75(10):4801-4805 (1978)). In someembodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9regions are used to characterize an OTU. In one embodiment, the V1, V2,and V3 regions are used to characterize an OTU. In another embodiment,the V3, V4, and V5 regions are used to characterize an OTU. In anotherembodiment, the V4 region is used to characterize an OTU. In oneembodiment of any of the compositions of the invention, the compositioncomprises one or more bacterial strains which are independentlycharacterized by, i.e., at least 95%, 96%, 97%, 98%, or 99% sequenceidentity to 16S rRNA sequences of the bacterial species recited in Table1B or Table 4. In one embodiment of any of the compositions of theinvention, the composition augments the growth of at least one type ofbacteria not detected in the probiotic or in the newborn'sgastrointestinal (GI) tract, skin, mouth, or any body site, prior toadministration.

In one embodiment of any of the compositions of the invention, saidcomposition comprises one or more bacterial strains which can be foundin a healthy vaginal microbiota from a pregnant woman in the thirdtrimester of pregnancy before or at the time of giving birth. In onespecific embodiment, the woman has not been administered antibioticcompounds within a certain period prior to isolation of bacteria(preferably, for at least one month prior to isolation of bacteria), hasbody mass index (BMI) between 18.5 and 24.9, does not have Group BStreptococcus (GBS), human immunodeficiency virus (HIV), Chlamydia,and/or sexually transmitted diseases, has vaginal pH less than 4.5, anddoes not have irritable bowel disease, Crohn's disease, ulcerativecolitis, irritable bowel syndrome, celiac disease, colorectal cancer ora family history of these diseases.

In one embodiment of any of the compositions of the invention, thecomposition comprises a buffering agent to adjust pH to the naturalvaginal pH at the time of labor or to a pH of 3.5 to 7. In oneembodiment of any of the compositions of the invention, the compositioncomprises an excipient or a carrier that optimizes the seeding of one ormore bacterial strains contained in the composition. In one embodimentof any of the compositions of the invention, the composition isformulated for storage in a frozen form. In one embodiment of any of thecompositions of the invention, said composition is a lyophilizedcomposition. Any of the compositions of the invention can contain, e.g.,live bacterial cells, conditionally lethal bacterial cells, inactivatedbacterial cells, killed bacterial cells, spores (e.g.,germination-competent spores), recombinant carrier strains, cellextract, or bacterially-derived products (natural or syntheticbacterially-derived products such as, e.g., bacterial antigens ormetabolic products). In one embodiment of any of the compositions of theinvention, the composition is formulated for delivery to the mouth,nose, and/or skin of the infant and/or for placing it on the maternalbreast and/or chest. In one embodiment of any of the compositions of theinvention, the composition is formulated for delivery by a routeselected from the group consisting of oral, topical, rectal, mucosal,sublingual, nasal, and via naso/oro-gastric gavage. In one embodiment ofany of the compositions of the invention, the composition is in a formof a liquid, foam, cream, spray, powder, or gel. In one embodiment ofany of the compositions of the invention, the composition comprises abuffering agent (e.g., sodium bicarbonate, infant formula or sterilizedhuman milk). In one embodiment of any of the compositions of theinvention, the composition comprises a prebiotic which stimulates growthand/or activity of one or more bacteria contained in the composition.Non-limiting examples of useful prebiotics include, e.g.,fructooligosaccharides (FOS), galactooligosaccharides (GOS), human milkoligosaccharides (HMO), Lacto-N-neotetraose, D-Tagatose,xylo-oligosaccharides (XOS), arabinoxylan-oligosaccharides (AXOS),N-acetylglucosamine, N-acetylgalactosamine, glucose, arabinose, maltose,lactose, sucrose, cellobiose, amino acids, alcohols, resistant starch(RS), and any mixtures thereof.

In a related aspect, the invention provides an absorbent material ordevice (e.g., gauze, sponge, or tampon) comprising any of thecompositions of the invention.

In another related aspect, the invention provides a method for restoringnormal microbiota in an infant delivered by Cesarean section or a methodfor restoring normal microbiota in a pre-term infant, said methodcomprising administering to said infant at the time of birth and/orwithin the first 4 months of life an effective amount of any of thecompositions of the invention.

In another related aspect, the invention provides a method for treatinga disease in a subject associated with the subject's delivery byCesarean section or with the subject's pre-term birth, said methodcomprising administering to said subject at the time of birth and/orwithin the first 4 months of life a therapeutically effective amount ofany of the compositions of the invention.

It is also contemplated that when used to treat various diseases, thecompositions and methods of the present invention can be utilized incombination with other therapeutic methods/agents suitable for the sameor similar diseases. Such other therapeutic methods/agents can beco-administered (simultaneously or sequentially) to generate additive orsynergistic effects. Suitable effective dosages for each agent may belowered due to the additive action or synergy.

In one embodiment of any of the above methods, the infant (or subject)is human. In one embodiment of any of the above methods, the infant is anewborn.

As also specified in the Background Section above, there is a great needin the art for a device and method for capturing, delivering, restoring,and/or improving normal microbiota from a mother to their newborn. Thepresent application addresses these and other needs by providing adevice and method of using the device for collecting and restoringviable, bioactive, and diverse normal natural microbiota, in infants,particularly those under non-naturally vaginal delivery, such as throughcesarean section, and/or those born in pre-term and/or prematureconditions.

In one embodiment, a medium sterile pad gauze (J&J, 7.6×7.6 cm) foldedlike a fan and then in half, was wet with sterile saline solution andintroduced in the maternal birth canal in the hour prior to theC-section, at the time antibiotics were administered. In certainembodiments, surgeon can extract the gauze prior to the procedure or anytime before the surgical procedure, incubating the gauze in the birthcanal for the time it takes to insert and extract, or for as long as 1hour.

The invention contemplates in a broad scope any carriers, container,and/or devices suitable for holding the maternal vaginal fluids and/ormicrobiota in a suitable condition that is optionally a sterilecondition. The invention also contemplates in a broad scope of devicesfor transferring, including but not limited to various absorbentmaterials in the form of gauze, sponge, tampon, etc., and/or via needle,tube, catheter, etc.

Certain embodiments of the invention can include a device to collectmaternal vaginal microbiota from the vaginal canal of a patient. Thedevice can have a housing forming a cavity, an absorbent removablematerial disposed within the cavity, and a deployment element disposedin the housing and movable along a length of the housing. The movementof the deployment element can displace the absorbent material out of thecavity and in to the vaginal canal.

Other embodiments of the device comprise an absorbent material that isnot a material such as, but not limited to, a menstrual pad, a tampon,or a urine pad. Further, the housing and the deployment element can besized to deliver the absorbent material within the lower ⅓ of thevaginal canal.

In a further embodiment, the absorbent. In other embodiments, the fulllength of the retrieval interface does not extend beyond the vaginalcanal. Further, the retrieval interface can have a retrieval elementcapturing the deployed absorbent material followed by removal of theabsorbent material from the vaginal canal. In some embodiments, thedeployment element acts as the retrieval element. In other embodiments,the absorbent material has the retrieval interface. In certainembodiments, the retrieval element captures the deployed absorbentmaterial by interacting with the retrieval interface. The retrievalelement can also be disposed within the housing and capture and returnthe deployed absorbent material to the cavity for the removal of theabsorbent material from the vaginal canal. Alternate embodiments have asecond housing forming a second cavity and the retrieval element isdisposed within the second housing and captures the deployed absorbentmaterial and returns the absorbent material to the second cavity for theremoval of the absorbent material from the vaginal canal.

In addition to the above embodiments, at least one of the housing, thecavity, the absorbent material and the deployment element can besterile. Also, the housing can include a seal around at least one of thecavity and the absorbent material to maintain sterility. The deploymentelement can break the seal when deploying the absorbent material.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the following description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further aspects of this invention may be better understoodby referring to the following description in conjunction with theaccompanying drawings, in which like numerals indicate like structuralelements and features in various figures. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

The drawing figures depict one or more implementations in accord withthe present teachings, by way of example only, not by way of limitation.In the figures, like reference numerals refer to the same or similarelements.

FIG. 1 . Restoring the maternal microbiota in C-section born infants.When antibiotics are administered 1 hour prior to the C-sectionprocedure, a gauze is inserted in the mothers' vagina, and extractedwhen the procedure starts. The gauze, kept in a sterile container, isused to swab the newborn as soon as possible after birth, starting withthe mouth, face and rest of the body.

FIG. 2 . Mean relative abundance of predominant bacteria (>1% in anysample) in mothers and babies of each delivery group, during the firstmonth of life of the baby. Taxa are reported at the lowest identifiablelevel.

FIGS. 3A-3C. Alpha diversity of baby oral (a), anal (b) and skin(forehead, arm and foot; c) samples, in relation to maternal sites,during the first 30 days of life of the baby. At birth, the bacterialdiversity in baby's mouth and anus (but not in the skin) is higher thanthe maternal vagina diversity (green area), but during the first week,it decreases to values below or similar to vaginal diversity, remaininglow during the period of strict lactation (about 3 months).

FIG. 4 . PCoA of the infant anal and skin microbiota during the firstmonth of life, in relation to maternal sites. The anal microbiota fromC-section-inoculated newborns cluster close to that in vaginallydelivered babies at day 1. Non inoculated C-section babies clusterseparately until the first week of life.

FIG. 5 . Lefse analyses depicting overrepresented taxa in the skinforehead of infants by mode of delivery at different times after birth

FIG. 6 . An example of a device to collect maternal vaginal microbiota.

FIGS. 7A-7C. Illustrates the deployment of the absorbent material.

FIGS. 8A-8H. Illustrates multiple examples of the deployment andretrieval devices.

FIGS. 9A-9H. Illustrates multiple examples of the deployment andplacement of the absorbent article.

FIGS. 10A-10E. Illustrates an example of a deployment and retrieval ofthe absorbent material into and removed from the vaginal canal.

FIGS. 10F-10G. Illustrate a variant example of a deployment andretrieval of the absorbent material into and removed from the vaginalcanal.

DETAILED DESCRIPTION

The present invention provides methods for restoring bioactivity anddiversity of normal microbiota in pre-term newborns and/or newbornsdelivered by Cesarean section and methods for treating (e.g., preventingor ameliorating) diseases associated with delivery by Cesarean sectionor pre-term birth comprising administering to said newborns at the timeof birth or shortly thereafter an effective amount of a vaginalmicrobiota inoculum obtained from the newborn's mother or a donor duringthe third trimester of pregnancy before or at the time of giving birthor an effective amount of a probiotic composition, wherein saidprobiotic composition (i) stimulates growth and/or activity of bacteriawhich are under-represented in microbiota of said newborn as compared tovaginally delivered full-term newborns, and/or (ii) inhibits growthand/or activity of bacteria which are over-represented in microbiota ofsaid newborn as compared to vaginally delivered full-term newborns. Alsoprovided are methods for diagnosing abnormal microbiota development in anewborn, comprising determining a relative abundance of one or morebacterial taxa in a microbiota sample obtained from said newborn

Definitions

As used herein, the terms “microbe” or “microorganism” encompass bothprokaryotic organisms including bacteria and archaea, and eukaryoticorganisms, including fungi, present in mammalian microbiota.

The terms “vaginal microbiota” or “vaginal flora” or “vaginalmicrobiome” are used interchangeably and refer to the microorganismsthat colonize the vagina.

The term “restoring normal microbiota” is used herein to refer torestoring microbiota of an infant (e.g., skin, oral, nasal,gastrointestinal, or any other mucosal microbiota) to the level ofbioactivity and diversity of corresponding microbiota of a healthyinfant delivered naturally, i.e., through labor and vaginal exposure.This may also be considered as normalizing the microbiota, populatingthe microbiota, populating normal microbiota, preventing the onset ofdysbiosis, or augmenting the growth of at least one type of bacteria inan infant. Preferably, such healthy naturally delivered “control” infantwas born to a mother who has not been administered antibiotic compoundswithin a certain period prior to the delivery (preferably, for at leastone month prior to the delivery), is not obese or overweight (preferablyhas body mass index (BMI) scores of below 25, most preferably between18.5 and 24.9), does not have Group B Streptococcus (GBS), humanimmunodeficiency virus (HIV), Chlamydia, and/or sexually transmitteddiseases, has vaginal pH less than 4.5, and does not have irritablebowel disease, Crohn's disease, ulcerative colitis, irritable bowelsyndrome, celiac disease, colorectal cancer and a family history ofthese diseases.

Specific taxa and changes in microbiota discussed herein can be detectedusing various methods, including without limitation quantitative PCR(qPCR) or high-throughput sequencing (e.g., shotgun metagenomesequencing) methods which detect over- and under-represented genes inthe total bacterial population (e.g., 454-sequencing for communityanalysis; screening of microbial 16S ribosomal RNAs (16S rRNA), etc.),or transcriptomic or proteomic studies that identify lost or gainedmicrobial transcripts or proteins within total bacterial populations, ormetabolomics. See, e.g., U.S. Patent Publication No. 2010/0074872;Eckburg et al., Science, 2005, 308:1635-8; Costello et al., Science,2009, 326:1694-7; Grice et al., Science, 2009, 324:1190-2; Li et al.,Nature, 2010, 464: 59-65; Bjursell et al., Journal of BiologicalChemistry, 2006, 281:36269-36279; Mahowald et al., PNAS, 2009,14:5859-5864; Wikoff et al., PNAS, 2009, 10:3698-3703.

As used herein, the term “16S rRNA sequencing” refers to the sequencingof 16S ribosomal RNA (rRNA) gene sequences by using primers such asuniversal primers and/or species-specific primers to identify thebacteria present in a sample. 16S rRNA genes contain both highlyconserved sites and hypervariable regions that can providespecies-specific signature sequences useful for identification ofbacteria. Such universal primers are well known in the art.

As used herein, the term “operational taxonomic unit” or “OTU” refers togroup of bacterial sequences that differ among each other in <97%identity. A “type” or a plurality of “types” of bacteria includes an OTUor a plurality of different OTUs, and also encompasses differences inspecies, genus, family or order of bacteria. The specific geneticsequence may be the 16S rRNA sequence or a portion of the 16S rRNAsequence or it may be a functionally conserved housekeeping gene foundbroadly across the eubacterial kingdom.

As used herein, the term “pre-term” as in pre-term newborn/infant orpre-term birth refers to any pre-term birth, including delivery beforethe 37-week gestation period. In some embodiments, pre-term includes anybirth on or before about 37 weeks. In some embodiments, delivery is fromabout 37 weeks to 39 weeks. In some embodiments, delivery is from about32 weeks to 37 weeks. In some embodiments, delivery is from about 32weeks to 28 weeks. In some embodiments, delivery is from about 23 weeksto 28 weeks. References to weeks of gestation include part weeks, suchthat a reference to 32 weeks includes both 32 weeks and 0 days through32 weeks and 6 days.

As used herein, the term “probiotic” refers to a substantially purebacteria (i.e., a single isolate, of, e.g., live bacterial cells,conditionally lethal bacterial cells, inactivated bacterial cells,killed bacterial cells, spores, recombinant carrier strains), or amixture of desired bacteria, bacteria components or bacterial extract,or bacterially-derived products (natural or syntheticbacterially-derived products such as, e.g., bacterial antigens ormetabolic products) and may also include any additional components thatcan be administered to a mammal. Such compositions are also referred toherein as a “bacterial inoculant.”

As used herein, the term “prebiotic” refers to an agent that increasesthe number and/or activity of one or more desired bacteria, enhancingtheir growth. Non-limiting examples of prebiotics useful in the methodsof the present invention include fructooligosaccharides (e.g.,oligofructose, inulin, inulin-type fructans), galactooligosaccharides,human milk oligosaccharides (HMO), Lacto-N-neotetraose, D-Tagatose,xylo-oligosaccharides (XOS), arabinoxylan-oligosaccharides (AXOS),N-acetylglucosamine, N-acetylgalactosamine, glucose, other five- andsix-carbon sugars (such as arabinose, maltose, lactose, sucrose,cellobiose, etc.), amino acids, alcohols, resistant starch (RS), andmixtures thereof. See, e.g., Ramirez-Farias et al., Br J Nutr (2008)4:1-10; Pool-Zobel and Sauer, J Nutr (2007), 137:2580S-2584S.

The terms “treat” or “treatment” of a state, disorder or conditioninclude: (1) preventing, delaying, or reducing the incidence and/orlikelihood of the appearance of at least one clinical or sub-clinicalsymptom of the state, disorder or condition developing in a subject thatmay be afflicted with or predisposed to the state, disorder or conditionbut does not yet experience or display clinical or subclinical symptomsof the state, disorder or condition; or (2) inhibiting the state,disorder or condition, i.e., arresting, reducing or delaying thedevelopment of the disease or a relapse thereof (in case of maintenancetreatment) or at least one clinical or sub-clinical symptom thereof; or(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms. The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

As used herein, the term “therapeutically effective amount” refers tothe amount of a microbiota inoculum or probiotic that, when administeredto a subject for treating (e.g., preventing or ameliorating) a state,disorder or condition, is sufficient to effect such treatment. The“therapeutically effective amount” will vary depending, e.g., on thebacteria or analogues administered as well as the disease and physicalconditions and responsiveness of the subject to be treated.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are generally regarded asphysiologically tolerable.

As used herein, the term “combination” of a microbiota inoculum orprobiotic and at least a second pharmaceutically active ingredient meansat least two, but any desired combination of compounds can be deliveredsimultaneously or sequentially (e.g., within a 24 hour period).

The terms “patient”, “individual”, “subject”, and “animal” are usedinterchangeably herein and refer to mammals, including, withoutlimitation, human and veterinary animals (e.g., cats, dogs, cows,horses, sheep, pigs, etc.) and experimental animal models. In apreferred embodiment, the subject (e.g., infant) is a human.

As used herein, the term “infant” refers to subjects from birth untilthe age when microbiome development is completed and encompasses newbornsubjects. For humans, “infant” refers to subjects from birth to 3 yearsof age. In some embodiments, an infant treated using any of the methodsor compositions of the invention is treated during the stage ofdevelopment relevant (or critical) to microbiome development.

As used herein, the term “stimulate” when used in connection with growthand/or activity of bacteria encompasses the term “enhance”.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Alternatively, the carrier can be a solid dosage formcarrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

The term “about” or “approximately” means within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, still morepreferably within 10%, and even more preferably within 5% of a givenvalue or range. The allowable variation encompassed by the term “about”or “approximately” depends on the particular system under study, and canbe readily appreciated by one of ordinary skill in the art.

The terms “a,” “an,” and “the” do not denote a limitation of quantity,but rather denote the presence of “at least one” of the referenced item.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of statistical analysis, molecularbiology (including recombinant techniques), microbiology, cell biology,and biochemistry, which are within the skill of the art. Such tools andtechniques are described in detail in e.g., Sambrook et al. (2001)Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005)Current Protocols in Molecular Biology. John Wiley and Sons, Inc.:Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in CellBiology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds.(2005) Current Protocols in Immunology, John Wiley and Sons, Inc.:Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols inMicrobiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al.eds. (2005) Current Protocols in Protein Science, John Wiley and Sons,Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols inPharmacology, John Wiley and Sons, Inc.: Hoboken, N.J. Additionaltechniques are explained, e.g., in U.S. Pat. No. 7,912,698 and U.S.Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437

Methods, Devices, and Compositions of the Invention

In the following detailed description, numerous specific details are setforth by way of examples in order to provide a thorough understanding ofthe relevant teachings. However, it should be apparent to those skilledin the art that the present teachings may be practiced without suchdetails. In other instances, well known methods, procedures, components,and/or circuitry have been described at a relatively high-level, withoutdetail, in order to avoid unnecessarily obscuring aspects of the presentteachings.

The vertical transmission of microbiota from mother to child duringlabor and birth is highly conserved in mammals, suggesting evolutionaryfitness (35). The present invention provides that major microbiotadeficiencies at birth in pre-term newborns or newborns delivered byCesarean section can be restored by the exposure to maternal (or thirdtrimester donor) vaginal microbiota or probiotics derived therefrom.

Microbiota donor subjects are generally of good health and havemicrobiota consistent with such good health. Often, the donor subjectshave not been administered antibiotic compounds within a certain periodprior to the collection of the microbiota (preferably, for at least onemonth prior to the collection of the microbiota). In certainembodiments, the donor subjects are not obese or overweight, and mayhave body mass index (BMI) scores of below 25, such as between 18.5 and24.9. In other embodiments, the donor subjects do not have irritablebowel disease, Crohn's disease, ulcerative coliti), irritable bowelsyndrome, celiac disease, colorectal cancer and a family history ofthese diseases. In other embodiments, donors have been screened forpathogens using standard techniques known to one in the art (e.g.nucleic acid testing, serological testing, antigen testing, culturingtechniques, enzymatic assays, assays of cell free fecal filtrateslooking for toxins on susceptible cell culture substrates).

In some embodiments, donors are also selected for the presence ofcertain genera and/or species that provide increased efficacy oftherapeutic compositions containing these genera or species. In otherembodiments, donors are preferred that produce relatively higherconcentrations of spores than other donors. In further embodiments,donors are preferred that provide spores having increased efficacy; thisincreased efficacy can be measured using in vitro or animal studies. Insome embodiments, the donor may be subjected to one or more pre-donationtreatments in order to reduce undesired material in the collectedmicrobiota, and/or increase desired spore populations.

It is advantageous to screen the health of the donor subject prior toand optionally, one or more times after, the collection of themicrobiota material. Such screening identifies donors carryingpathogenic materials such as viruses (e.g., HIV, hepatitis, polio) andpathogenic bacteria. Post-collection, donors are screened about oneweek, two weeks, three weeks, one month, two months, three months, sixmonths, one year or more than one year, and the frequency of suchscreening may be daily, weekly, bi-weekly, monthly, bi-monthly,semi-yearly or yearly. Donors that are screened and do not testpositive, either before or after donation or both, are considered“validated” donors.

Methods for collection and preparation of vaginal microbiota are wellknown in the art. The vaginal microbiota can be stored, e.g., as frozenor lyophilized preparations (said lyophilized preparations can bereconstituted prior to the administration to the infant) or can beprocessed to isolate desired bacteria as single or mixed cultures andthen stored. Vaginal microbiota and probiotics can be administered invarious forms, including but not limited to, solid (e.g., powder),liquid, gel, cream, spray, foam, etc. The invention contemplates the useof various carriers, containers, and devices suitable for holding thevaginal microbiota in a suitable condition. For vaginal microbiota andlive bacteria probiotic preparations, the carrier should preferablycontain an ingredient that promotes viability of the bacteria duringstorage. The formulations can include added ingredients to improvepalatability, improve shelf-life, impart nutritional benefits, and thelike. In certain embodiments, the microbiota and probiotic preparationsused in the methods of the invention comprise a buffering agent toadjust pH to the natural vaginal pH at the time of labor (pH of 3.5-7)or to a pH (3.5-7) that optimizes the seeding of the transferredmicrobiota.

The invention also contemplates in a broad scope any means fortransferring, including but not limited to various absorbent materials(e.g., in the form of gauze, sponge, tampon, etc.), and/or needle, tube,catheter, etc. The vaginal microbiota can be transferred to absorbentmaterial or device, e.g., by introducing said absorbent material ordevice in vagina prior to the birth or at the time of Cesarean section(e.g., for at least 5 minutes).

The invention provides that the collected vaginal microbiota orprobiotics can be administered to any body part colonized in thenewborns, including but not limited to, mouth, nasal mucosa, skin, etc.Alternatively (or in addition), vaginal microbiota or probiotics can beplaced on the maternal breast and/or chest. Non-limiting examples ofsuitable routes of administration of vaginal microbiota and probioticsinclude oral (e.g., swabbing or via feeding tube or baby bottle),topical, rectal (e.g., by enema), mucosal, sublingual, nasal, and vianaso/oro-gastric gavage. If a reproducible and measured dose is desired,the bacteria can be administered by a rumen cannula.

The dosages of the microbiota inoculum or probiotic administered in themethods of the invention will vary widely, depending upon the newborn'sphysical parameters, the frequency of administration, the manner ofadministration, the clearance rate, and the like. The initial dose maybe larger, and might be followed by smaller maintenance doses. The dosemay be administered as infrequently as weekly or biweekly, orfractionated into smaller doses and administered daily, semi-weekly,etc., to maintain an effective dosage level. It is contemplated that avariety of doses will be effective to achieve colonization, e.g. 10⁶,10⁷, 10⁸, 10⁹, and 10¹⁰ CFU for example, can be administered in a singledose. Lower doses can also be effective, e.g., 10⁴, and 10⁵ CFU.

Bacterial strains administered in probiotics according to the methods ofthe present invention can comprise live bacteria. One or severaldifferent bacterial inoculants can be administered simultaneously orsequentially (including administering at different times). Such bacteriacan be isolated from vaginal microbiota and grown in culture using knowntechniques. However, many bacterial species are very difficult toculture and administration of others may lead to various undesirableside-effects. The present invention also comprises administering“bacterial analogues”, such as recombinant carrier strains expressingone or more heterologous genes derived from the relevant bacterialspecies. The use of such recombinant bacteria may allow the use of lowertherapeutic amounts due to higher protein expression. In certainembodiments, spores, killed bacterial cells and bacterial cell extractscan be utilized as the probiotics of the invention (see, e.g., Round etal., Proc. Natl. Acad. Sci. USA, 2010, 107: 12204). Bacteria in thecompositions of the invention can be from one or more different speciesand can be, e.g., in the form of live bacterial cells, conditionallylethal bacterial cells, inactivated bacterial cells, killed bacterialcells, spores (e.g., germination-competent spores), recombinant carrierstrains, cell extract, or bacterially-derived products (natural orsynthetic bacterially-derived products such as, e.g., bacterial antigensor metabolic products). In one specific embodiment, the compositioncomprises at least two different bacterial strains.

In one specific embodiment, the composition comprises bacteria from atleast two different bacterial species. In some embodiments, thecompositions comprise bacteria from at least 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 200, 500, or 1000 different bacterial species.

In some embodiments, the composition comprises bacteria from at leastone of the taxa provided in Table 1A. In some embodiments, thecomposition comprises bacteria from at least 2, 3, 4, 5, 6, 7, 8, 9, 10,20, or 50 different taxa in Table 1A. In some embodiments, onlynonpathogenic species within the taxa qualify for use in thecompositions or methods herein.

TABLE 1A Taxa for Inclusion in Compositions and Methods or forMicrobiota Monitoring Family Neisseriacea Genera AcinetobacterActinomyces Aerococcus Alloscardovia Anaerococcus AnaerostipesAnoxybacillus Asticcacaulis Atopobium Bacillus BacteroidesBifidobacterium Blastomonas Bradyrhizobium Brevibacterium CampylobacterClavibacter Cloacibacterium Clostridium Comamonas CorynebacteriumDermabacter Devriesea Dialister Dorea Enterococcus EremococcusErysipelatoclostridium Escherichia Ezakiella Facklamia FastidiosipilaFenollaria Finegoldia Fusobacterium Gardnerella Gemella HaemophilusHelcococcus Herbaspirillum Jonquetella Kocuria Kytococcus LactobacillusLactococcus Leptotrichia Levyella Megasphaera MethylobacillusMethylobacterium Micrococcus Mobiluncus Mycoplasma NegativicoccusNovosphingobium Oceanitalea Parabacteroides Paracoccus ParvimonasPelistega Peptococcus Peptoniphilus Peptostreptococcus PolaromonasPorphyromonas Prevotella Pseudoclavibacter Pseudomonas RothiaRubrobacter Ruminococcus Sneathia Sphingobium SphingopyxisStaphylococcus Stenotrophomonas Streptococcus Sutterella UreaplasmaVaribaculum Veillonella Veillonella 1-68 BacteroidaceaPorphyromonadaceae Coriobacteriales Clostridiaceae Bacteriodales

In some embodiments, the composition comprises bacteria from at leastone of the species provided in Table 1B. In some embodiments, thecomposition comprises bacteria from at least 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, 100, or 150 different bacterial species in Table 1B. In someembodiments, at least some of the bacteria chosen from Table 1B are indifferent genera, including, but not limited to 2, 3, 4, 5, 6, 7, 8, 9,10, 20, or 50 different genera. In other embodiments, at least some ofthe bacteria chosen from Table 1B are in the same genera.

TABLE 1B Species for Inclusion in Compositions and Methods or forMicrobiota Monitoring Acinetobacter baumannii Acinetobacter gerneriAcinetobacter johnsonii Acinetobacter radioresistens Acinetobacterschindleri Acinetobacter seifertii Acinetobacter variabilis Actinomyceseuropaeus Actinomyces neuii Aerococcus christensenii Alloscardoviaomnicolens Anaerococcus lactolyticus Anaerococcus murdochii Anaerococcusobesiensis Anaerococcus octavius Anaerococcus prevotii Anaerococcusprovenciensis Anaerococcus vaginalis Anaerostipes hadrus Anoxybacillusflavithermus Asticcacaulis excentricus Atopobium deltae Atopobiumvaginae Bacillus vireti Bacteroides finegoldii Bacteroides vulgatusBifidobacterium breve, Bifidobacterium Blastomonas natatoriaBradyrhizobium lupini pseudolongum Brevibacterium paucivoransCampylobacter coli Campylobacter hominis Campylobacter ureolyticusClavibacter michiganensis Cloacibacterium rupense Clostridiumclostridioforme Clostridium perfringens Comamonas serinivorans Comamonastestosterone Corynebacterium Corynebacterium appendicis amycolatumCorynebacterium Corynebacterium Corynebacterium canis argentoratenseaurimucosum Corynebacterium casei Corynebacterium coyleaeCorynebacterium freneyi Corynebacterium imitans Corynebacterium jeikeiumCorynebacterium kroppenstedtii Corynebacterium lactis Corynebacteriummatruchotii Corynebacterium mycetoides Corynebacterium mycetoidesCorynebacterium pilbarense Corynebacterium pyruviciproducensCorynebacterium Corynebacterium striatum Corynebacterium spheniscorumterpenotabidum Corynebacterium thomssenii CorynebacteriumCorynebacterium tuscaniense tuberculostearicum Dermabacter hominisDevriesea agamarum Dialister micraerophilus Dialister propionicifaciensDialister succinatiphilus Dorea longicatena Enterococcus hiraeEremococcus coleocola Erysipelatoclostridium ramosum Escherichiamarmotae Ezakiella peruensis Facklamia hominis Facklamia ignavaFastidiosipila sanguinis Fenollaria massiliensis Finegoldia magnaFusobacterium equinum Fusobacterium nucleatum FusobacteriumFusobacterium simiae Gardnerella vaginalis periodonticum Gemellaasaccharolytica, Gemella taiwanensis Haemophilus pittmaniae Helcococcussueciensis Herbaspirillum Jonquetella anthropic chlorophenolicum Kocuriaflava strain HO- Kocuria kristinae strain DSM Kytococcus schroeteristrain 9041 20032 Muenster 2000 Lactobacillus coleohominis Lactobacilluscrispatus Lactobacillus hominis Lactobacillus iners Lactobacillusintestinalis Lactobacillus jensenii Lactobacillus psittaci Lactobacillusreuteri Lactobacillus rodentium Lactococcus lactis Levyella massiliensisMethylobacillus flagellates Methylobacterium aerolatum MethylobacteriumMicrococcus aloeverae strain phyllostachyos AE-6 Mobiluncus curtisiiMycoplasma hominis Negativicoccus succinicivorans Oceanitaleananhaiensis Parabacteroides faecis Parabacteroides merdae Paracoccuscommunis Parvimonas micra Pelistega indica Peptococcus nigerPeptoniphilus coxii Peptoniphilus duerdenii Peptoniphilus grossensisPeptoniphilus koenoeneniae Peptoniphilus lacrimalis Peptoniphilus obesistrain Peptoniphilus senegalensis Peptoniphilus tyrrelliae ph1Peptostreptococcus Polaromonas sp Porphyromonas bennonis anaerobiusPorphyromonas somerae Porphyromonas uenonis Prevotella amnii Prevotellabergensis Prevotella bivia Prevotella buccalis Prevotella copriPrevotella corporis Prevotella disiens Prevotella timonensisPseudoclavibacter bifida Pseudomonas brenneri strain IAM 14848Pseudomonas helmanticensis Pseudomonas lini Pseudomonas syringae Rothiaamarae strain J18 Rothia mucilaginosa Rubrobacter calidifluminisRuminococcus bromii Ruminococcus gnavus Sneathia sanguinegensSphingobium yanoikuyae Sphingopyxis Staphylococcus carnosusmacrogoltabida Staphylococcus chromogenes Staphylococcus petrasiiStaphylococcus pseudintermedius Staphylococcus StenotrophomonasStreptococcus agalactiae saprophyticus maltophilia Streptococcusanginosus Streptococcus constellatus Streptococcus dentisaniStreptococcus lactarius Streptococcus thermophiles Sutterellastercoricanis Ureaplasma urealyticum Varibaculum cambriense Veillonelladispar Veillonella ratti Bacteriodales S24-7

Within a given composition, different bacterial strains can be containedin equal amounts (even combination) or in various proportions (unevencombinations) needed for achieving the maximal biological activity. Forexample, in a bacterial composition with two bacterial strains, thestrains may be present in from a 1:10,000 ratio to a 1:1 ratio, from a1:10,000 ratio to a 1:1,000 ratio, from a 1:1,000 ratio to a 1:100ratio, from a 1:100 ratio to a 1:50 ratio, from a 1:50 ratio to a 1:20ratio, from a 1:20 ratio to a 1:10 ratio, from a 1:10 ratio to a 1:1ratio. For bacterial compositions comprising at least three bacterialstrains, the ratio of strains may be chosen pairwise from ratios forbacterial compositions with two strains. For example, in a bacterialcomposition comprising bacterial strains A, B, and C, at least one ofthe ratios between strain A and B, the ratio between strain B and C, andthe ratio between strain A and C may be chosen, independently, from thepairwise combinations above. In one embodiment, two or more bacterialstrains in the composition produce synergistic activity. In one specificembodiment, the invention encompasses administering two or morebacteria-containing compositions to the same subject. Such compositionscan be administered simultaneously or sequentially.

Spores used in the compositions of the invention can me isolated, forexample, by solvent treatments (e.g., using partially miscible, fullymiscible or an immiscible solvent), chromatographic treatments (e.g.,using hydrophobic interaction chromatography (HIC) or an affinitychromatography), mechanical treatments (e.g., blending, mixing, shaking,vortexing, impact pulverization, and sonication), filtration treatments,thermal treatments (e.g., 30 seconds in a 100° C. environment followedby 10 minutes in a 50° C.), irradiation treatments (e.g., with ionizingradiation, typically gamma irradiation, ultraviolet irradiation orelectron beam irradiation provided at an energy level sufficient to killpathogenic materials while not substantially damaging the desired sporepopulations), centrifugation and density separation treatments (e.g.,using density or mobility gradients or cushions (e.g., step cushions),such as, e.g., CsCl, Percoll, Ficoll, Nycodenz, Histodenz or sucrosegradients). It is generally desirable to retain the spore populationsunder non-germinating and non-growth promoting conditions and media, inorder to minimize the growth of pathogenic bacteria present in the sporepopulations and to minimize the germination of spores into vegetativebacterial cells.

The compositions of the invention can comprise a carrier and/orexcipient. While it is possible to use a bacterial inoculant or compoundof the present invention for therapy as is, it may be preferable toadminister it in a pharmaceutical formulation, e.g., in admixture with asuitable pharmaceutical excipient and/or carrier selected with regard tothe intended route of administration and standard pharmaceuticalpractice. The excipient and/or carrier must be “acceptable” in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient thereof. Acceptable excipients andcarriers for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington: The Science and Practiceof Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005).The choice of pharmaceutical excipient and carrier can be selected withregard to the intended route of administration and standardpharmaceutical practice. Oral formulations readily accommodateadditional mixtures, such as, e.g., milk, yogurt, and infant formula.Solid dosage forms for oral administration can also be used and caninclude, e.g., capsules, tablets, caplets, pills, troches, lozenges,powders, and granules. Non-limiting examples of suitable excipientsinclude, e.g., diluents, buffering agents (e.g., sodium bicarbonate,infant formula, sterilized human milk, or other agents which allowbacteria to survive and grow [e.g., survive in the acidic environment ofthe stomach and to grow in the intestinal environment]), preservatives,stabilizers, binders, compaction agents, lubricants, dispersionenhancers, disintegration agents, antioxidants, flavoring agents,sweeteners, and coloring agents. Those of relevant skill in the art arewell able to prepare suitable solutions.

The bacteria-containing formulations of the invention may comprise oneor more prebiotics which promote growth and/or activity of the bacteriain the formulation.

Non-limiting examples of prebiotic agents useful in the methods of thepresent invention include fructooligosaccharides (e.g., oligofructose,inulin, inulin-type fructans), galactooligosaccharides, human milkoligosaccharides (HMO), Lacto-N-neotetraose, D-Tagatose,xylo-oligosaccharides (XOS), arabinoxylan-oligosaccharides (AXOS),N-acetylglucosamine, N-acetylgalactosamine, glucose, other five andsix-carbon sugars (e.g., arabinose, maltose, lactose, sucrose,cellobiose, etc.), amino acids, alcohols, resistant starch (RS), andmixtures thereof. Additional prebiotic agents can be selected based onthe knowledge of particular bacteria.

Methods for producing bacterial compositions of the invention mayinclude three main processing steps, combined with one or more mixingsteps. The steps are: organism banking, organism production, andpreservation. For banking, the strains included in the bacterialcompositions of the invention may be (1) isolated directly from aspecimen or taken from a banked stock, (2) optionally cultured on anutrient agar or broth that supports growth to generate viable biomass,and (3) the biomass optionally preserved in multiple aliquots inlong-term storage. The bacterial suspension can be freeze-dried to apowder and titrated. After drying, the powder may be blended to anappropriate potency, and mixed with other cultures and/or a filler suchas microcrystalline cellulose for consistency and ease of handling, andthe bacterial composition formulated as provided herein.

Additional methods include methods of evaluating the microbiotapopulation in a subject or diagnosing an abnormal microbiotadevelopment. Methods include monitoring the infant's microbiota afterthe administration of the vaginal microbiota inoculum or probiotic by:(a) determining a relative abundance of one or more bacterial taxa in amicrobiota sample obtained from the infant, and (b) comparing therelative abundance(s) determined in step (a) to (i) a predeterminedstandard value or (ii) to the abundance(s) of the same taxa in a controlsubject or (iii) to the average value of abundances of the same taxa inseveral control subjects. The newborn's sample may be isolated fromfeces, skin, oral mucosa, conjunctive mucosa, or nasal mucosa. It may becompared to a control subject who is a vaginally delivered full-termhealthy infant. The control subject may be born to a mother who has notbeen administered antibiotic compounds within a certain period prior togiving birth (preferably, for at least one month prior to giving birth),has body mass index (BMI) between 18.5 and 24.9, and does not haveirritable bowel disease, Crohn's disease, ulcerative colitis, irritablebowel syndrome, celiac disease, colorectal cancer, and a family historyof these diseases.

The relative abundance of the taxa may comprise a method selected fromthe group consisting of quantitative polymerase chain reaction (qPCR),sequencing of bacterial 16S rRNA, shotgun metagenome sequencing, andmetabolomics.

In accordance with the present invention there may be numerous tools andtechniques within the skill of the art, such as those commonly used inmolecular biology, pharmacology, and microbiology. Such tools andtechniques are described in detail in e.g., Sambrook et al. (2001)Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005)Current Protocols in Molecular Biology. John Wiley and Sons, Inc.:Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in CellBiology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds.(2005) Current Protocols in Immunology, John Wiley and Sons, Inc.:Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols inMicrobiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al.eds. (2005) Current Protocols in Protein Science, John Wiley and Sons,Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols inPharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.

In some non-limiting embodiments, the compositions of the invention areformulated as pharmaceutical preparations for oral, topical, nasal,rectal, mucosal, sublingual, or nasal administration. In someembodiments, the formulation is a slow release formulation. In someembodiments, the compositions are formulated as medical foods,nutritional or dietary supplements, food products or beverage products

The invention also contemplates in a broad scope any means fortransferring, including but not limited to, various absorbent materials(e.g., in the form of gauze, sponge, tampon, etc.), and/or needle, tube,catheter, etc. The vaginal microbiota can be transferred to an absorbentmaterial or device, e.g., by introducing said absorbent material ordevice into the vagina prior to the birth or at the time of Cesareansection (e.g., for at least 1 second, at least 3 seconds, at least 5seconds, at least 10 seconds, at least 20 seconds, at least 30 seconds,at least 40 seconds, at least 50 seconds, at least 1 minute, at least 2minutes, at least 3 minutes, at least 4 minutes, or at least 5 minutes).

The examples of devices and methods of using the devices of the presentinvention enable a physician to collect and restore the maternalmicrobiota in C-section born infants. This primary example isillustrated in FIG. 1 . Prior to the C-section procedure, the absorbentmaterial or device is inserted in the mother's vagina and extracted whenthe procedure starts. This is illustrated in step 1. The absorbentmaterial or device is kept in a sterile container, for example, eitherin a separate container or in the original housing as described below.The absorbent material or device with the collected vaginal microbiotais stored during the delivery procedure. See, step 2. As soon aspossible after birth the absorbent material or device is retrieved andis used to swab the infant, starting with the mouth, face, and rest ofthe body. This is illustrated in step 3.

Storage, as used in some examples, can involve differing ranges oftemperatures depending on the when the collected microbiota is used. Insome circumstances when the microbiota can be swabbed onto the infantwithin a short amount of time after collection (e.g., 1 to 2 hours) theabsorbent material or device with the collected vaginal microbiota canbe stored at a temperature between the vaginal temperature and roomtemperature. For longer term storage, typically for future use (i.e. notto be used with child being born at the time of collection), thematernal microbiota can be stored at temperatures that will preserve thematernal microbiota for long periods. In some examples, the storagetemperature can be below freezing. These freezing temperatures can rangefrom just below the freezing point to deep freezing tens or even hundreddegrees below the freezing point. The temperature used can be dictatedby the need for long term preservation and harmful effects to the storedmaternal microbiota. In certain embodiments, the microbiota is stored atultralow temperatures. In additional embodiments, the microbiota isstored in a media containing 15% glycerol. In certain embodiments, the15% glycerol helps to preserve cell viability.

FIG. 6 illustrates a device 100 to collect the maternal microbiota,which includes a housing 102 designed to be inserted into the vagina ofthe expecting mother prior to delivery of the infant. The housing 102has a proximal end 104 and a distal end 106 and can enclose a cavity108. The cavity 108 can hold an absorbent material 200 which can bedeployed into the vagina to collect the maternal vaginal fluids, and/orthe maternal vaginal microbiota. The distance between the proximal anddistal ends 104,106 can describe a housing length λ.

In an embodiment, the proximal end 104 of the housing 102 can have adeployment/retrieval element 300 to deploy the absorbent material 200once the housing 102 is placed in the vagina, and it can be deployed outof the distal end 106. In certain embodiments, the deployment/retrievalelement 300 is slidable along at least a part of the housing length λ.This includes the ability to be slidable within the cavity 108. Thedeployment/retrieval element 300 can also be removable from the housing102.

One embodiment, the deployment/retrieval element 300 can deploy theabsorbent material 200 followed by the removal of the housing 102. Theabsorbent material 200 can then be removed with a second device, havinga second housing/cavity or by a medical professional (e.g., surgeon)prior to the delivery of the baby. In other aspects, the absorbentmaterial 200 is deployed and after a predetermined amount of time, theabsorbent material 200 is retrieved by the same device 100. In certainembodiments, the device 100 can remain inside the vagina for theduration of the collection of the maternal vaginal fluids, and/or thematernal vaginal microbiota or can be removed after deployment of theabsorbent material 200 and reintroduced to retrieve the absorbentmaterial 200.

The above situations lead to different embodiment of the device 100 andits elements. In an example, the one or more of the device 100, housing102, absorbent material 200, deployment/retrieval element 300 and cavity108, and portions thereof, can be sterilized. This can be as simple assterilizing the device in an autoclave prior to use or factorysterilization and sterile packaging. Other embodiments can include aseal 112 that seals the cavity 108, and/or the proximal 104 and distal106 ends. In certain embodiments, the seal 112 makes just those sections(e.g., 108, 104, and 106) sterile environments.

In FIG. 7A it is illustrated that the cavity 108 containing theabsorbent material 200 is sealed 112 at both ends. The cavity 108 can bea sterile environment or the absorbent material 200 can be in sterilewrapping and the seals 112 are surrounding the absorbent material 200.Once the housing 102 is inserted into the vagina the absorbent material200 can be deployed. The act of deployment can break the seal(s) 112 sothe absorbent material 200 is sterile as it enters the vagina. This canbe caused by the pressure of the absorbent material 200 against theseal(s) 112 as it is being deployed or a feature of the deploymentelement 300 that breaks the seal 112 prior to, or as, the absorbentmaterial 200 is being deployed. FIG. 7B illustrates this deployment andFIG. 7C illustrates the fully deployed absorbent material 200 and thepartial removal of the housing 102. In some examples, the deliverydevice 100 can be designed to deliver the absorbent material 200 nofarther than the lower third of the vaginal canal V.

There are many embodiments of the deployment element 300. As illustratedin FIGS. 8A-8B, the deployment element 300 can be as simple as aplunger-type device 302 which is partially exposed outside the distalend 106. In certain embodiments, as the user pushes on the plunger 302,it is moved into the cavity 108 to displace the absorbent material 200and forces it out the proximal end 104. Other embodiments can have thedeployment element 300 also retrieve the absorbent material 200. Forexample, deployment/retrieval element 304 can be tethered to theabsorbent material 200 so that operation of the element 304 can pull theabsorbent material 200 back into the cavity for retrieval and removalfrom the vagina. As illustrated in FIGS. 8C and 8D, this can be tether305.

Other deployment and retrieval elements 306 can be unattached to theabsorbent material 200 but designed to grab or capture the materialthrough the use of a reattachable interface 307. The reattachableinterface can be, for example, mating hook and loop material on each ofthe element 306 and the absorbent material 200, attractive magnets, or aparticular male/female locking point. See FIGS. 8E and 8F.

FIGS. 8G and 8H illustrate further deployment and retrieval elements308. These retrieval devices can have a capture adapter 309 designed toretrieve the absorbent material 200 independent of the structure of theabsorbent material 200. Non-limiting embodiments can be hooks, anexpandable “claw”, and suction.

Note that the deployment and retrieval elements can be in the samehousing 102 or from separate devices. In addition, combinations ofdeployment and retrieval embodiments can be interchanged and/or can bebased on the user performing the insertion and removal. For example, ifthe user is the expectant mother, simplified embodiments of the devicesdescribed above and below can be used. If a medical professional isassisting, more complex devices can be deployed. In certain embodiments,the absorbent material can lack a retrieval interface (tether,monofilament plastic line, reattachable interface, etc.) and only aretrieval device can capture and remove the absorbent material.

In further embodiments, the deployment and retrieval elements can have alength L (as illustrated in FIGS. 8D and 8H) and the full length ofwhich can extend outside the vagina. Other embodiments of deployment andretrieval elements can have a full length L that does not extend outsidethe vagina or vaginal cavity V.

Additionally, the deployment and retrieval elements can be separateelements using the same housing. Thus, the user deploys the absorbentmaterial 200, removes the deployment element from the housing andreplaces it with a retrieval element in the same housing. The switch ofelements can be performed while the housing is outside or inside thevaginal canal V.

Turning now to the absorbent material 200 it can be in a form of a foam,a solution, liquid, cream, or gel. Alternately or additionally, theabsorbent material 200 can be in the form of a gauze, sponge, or tamponlike materials. Unlike a tampon, examples of the absorbent material 200can be designed not to absorb large or significant quantities of fluid.The absorbent material's 200 purpose is to sample small amount/collectthe maternal vaginal fluids, and/or the maternal vaginal microbiota. Theabsorbent material 200 is not designed to stop vaginal discharges.

Absorbency can be measured in units of grams of fluid over grams ofmaterial (g/g). As an example, tampons are approximately 7 grams inweight and can absorb between 6 and 18 grams of fluid, depending ontheir construction (light flow to ultra absorbency), leading toabsorbency units of 0.85 to 2.57 g/g of absorbency. Surgical sponges canabsorb up to 12.29 g/g. The absorbent material can, in one example, havethe absorbency of sterile gauze used for wound dressing. In otherembodiment, the absorbent material can have the absorbency less thanthat of a light flow tampon or can have an absorbency of less than 1.25g/g or less than 0.85 g/g.

The natural birth of a baby involves the infant passing through thebirth canal prior to delivery. Here a large portion of the baby's skinsurface is in contact with the birth canal for what may be an extendedperiod of time. In this passing, the infant contacts the maternalvaginal fluids, and/or the maternal vaginal microbiota as noted above.The materials and sampling techniques of this invention attempt to mimicat least partially this “trip”.

Thus, collection of the maternal vaginal fluids, and/or the maternalvaginal microbiota may also include a tissue sampling component. Thecollection the maternal vaginal fluids, and/or the maternal vaginalmicrobiota can entail the retrieval of a small amount of tissue from thewalls of the vagina, this can be as small as just a number of cellsloughed off the vaginal wall.

The structure of the absorbent material 200 can be selected orengineered as such to have a particular pore size that is conducive tocollecting small amounts of vaginal fluid, or a surface roughness tofacilitate cell collection. The absorbent material 200 can be furtherdesigned or selected to be expandable or swell once deployed.

FIGS. 9A-9H illustrate different embodiments of the absorbent material200. FIG. 9A illustrates absorbent material 200 designed to swell oncereleased from the housing 102. This can be based on the absorption offluids from the vaginal canal V. The swelling, in certain embodiments,can be enough to fill a portion of the vaginal canal V and have theabsorbent material 200 contact the walls of the vaginal canal V.

FIG. 9B illustrates the use of a foam or gel 202, which can be designedto swell and even increase its rigidity to allow the whole foam/gel tobe retrieved in one collection. In addition, given their nature they canmake contact with a large surface area of the birth canal. Materials canalso be designed with the same properties, whether structurally ormechanically.

The absorbent material 204 can be a standard material but folded in sucha way to one or both of maximize compaction to be as compact as possiblein the device 100 prior to vaginal delivery and then to expand or unfoldto achieve the maximum surface area contact with the vaginal walls. Theabsorbent material 204 can be folded or twisted accordingly.Non-limiting examples can be a fan fold (FIG. 9C) and an “umbrella” typefold (FIG. 9D). A “fan fold” is taking the absorbent material 204 andmaking a first fold a small distance from the edge of the material. Theabsorbent material 204 is then flipped over and a second fold is made,approximately the same size as the first fold. The material 204 isflipped over again to its original side and a third fold is made,approximately the same size as the first and second folds. This processis repeated until there is no more material to fold. The small distance,in one example, can be an even fraction of the entire length of thematerial, so as to make each fold of the fan approximately the samesize.

Additionally, the material can be designed to have a “shape memory”where it can be folded to be inserted into the cavity 106, but oncedeployed, it can take a performed shape. FIGS. 9E and 9F illustrate ashape memory absorbent device 206, first within the housing 102, andcompressed into a small profile shape. Next, in FIG. 9F, the shapememory absorbent device 206 can be deployed into the vaginal canal V andexpanded to contact the vaginal walls.

A yet another embodiment, the absorbent material 208 allows it tomechanically expand. Like the shape memory absorbent device 206, themechanically expanding absorbent material 208 has a compact shape in thehousing 102 (as seen in longitudinal cross-section, FIG. 9G) and canthen expand, using mechanical elements (e.g. a spring) 209, to take itsexpanded shape (as seen in longitudinal cross-section, FIG. 9H).

The absorbent material 200, 202, 204, 206, 208 can be appropriatenatural or synthetic material that functions to capture the vaginalfluids and microbiota. These materials include cotton, sponge, andcertain polymers, for example polypropylene/polyglycolic acid (PGA),polypropylene/polylactic acid (PLA), dacron, polyester. In addition,nitinol can be used for shape memory examples, as described above.Further, these materials can be spun, woven, knitted, braded, ormanufactured unwoven to provide the necessary surface area for optimalcollection. In addition, the absorbent material can be textured if skincell samples are deemed appropriate for collection as well. However, inat least one example, the material is not designed to absorb enoughfluid as to act as a menstrual pad, tampon, or urine pad.

In certain embodiments, the absorbent material 200, 202, 204, 206, 208can have a retrieval device 305, similar to a tether or anchor (asillustrated in FIG. 8D) that facilitates its retrieval by either adevice 100 or a medical professional (e.g., surgeon). The tether 305 canremain internal to the vaginal opening or can extend outside the openingso further insertions into the vagina are not required to retrieve theabsorbent material 200, 202, 204, 206, 208.

Retrieval and storage are another aspect of the invention. Embodimentscan include non-device retrieval, in which the user (surgeon, expectantmother, or nurse) removes the absorbent material 200, or retrieval canbe effected by the same insertion device 100 or a retrieval device. Anindependent retrieval device can be the same as described above in FIGS.8C-8H. Once retrieved the absorbent material 200 can be used to swab theinfant or can be stored for later use.

Storage, as used herein, can mean relatively soon after retrieval, at atime soon after the delivery of the newborn, and/or at a date in thefuture. Later or delayed use of the absorbent material 200 leads toexamples where the absorbent material 200 can be stored in a container.The sterile container can be a separate container from thedeployment/retrieval element 300 or the absorbent material 200 can bedrawn back into the device 100, or a second retrieval/storage device.FIG. 1 , step 2 illustrates the use of a separate container, which isnot used for deployment or retrieval.

As above, storage, for both time and temperature, is dependent on theuse. When used “contemporaneously” with the birth of the infant, theadsorbent material is deployed in the mother and removed soon before thec-section. How soon is dependent on the surgical team, but likely within10 minutes of the start of the procedure. The C-section can take from10-15 minutes to deliver the infant. After delivery, the infant can beswabbed with the absorbent material. Thus, in an example,“contemporaneous” use can be within an hour of removal of the absorbentmaterial from the vagina. This can be an approximately 1-2 hour window.Longer storage can be contemplated in days, months or years, dependingon when the microbiota sample is taken.

A mother may take the sample early if she is aware of circumstances thatmay cause a change in her natural microbiota before childbirth, butwithin the pregnancy cycle. A mother may also take a long term orredundant sample for future use with other infants to be born at afuture date.

FIGS. 10A to 10E illustrate an example where the absorbent material 200is retrieved into a deployment/retrieval device 400. This device can besame, or different from, the deployment device described above. Theillustrated example uses a tethered version of the absorbent article,but any of the above described retrieval device methods can be used.

FIG. 10A illustrates the loaded deployment/retrieval device 400 having ahousing 402 and a deployment/retrieval element 404 tethered to theabsorbent material 200. The absorbent material 200 can be any of theembodiments discussed above. A portion of the housing 402 is a sealedcavity 408. The cavity 408 is sealed by sterile seals 412 to maintainthe sterility of the absorbent material 200 prior to deployment.

FIGS. 10B and 10C illustrate partial deployment of the absorbentmaterial 200 and its in situ positioning expanded to full size,respectively. In FIG. 10B, at least the front seal 412A is broken as theabsorbent material 200 is displaced out of the cavity 408 and in to thevaginal canal V. The rear seals 412B may or may not be compromised whenthe deployment/retrieval element 404 is displaced to deploy theabsorbent material 200. This embodiment illustrates a tether 405disposed through the deployment/retrieval element 404 so that as theabsorbent material 200 is deployed so is the tether 405. However, thetether 405 is designed as such that it extends either a sufficientlength down the vaginal canal V or extends outside the vagina tofacilitate its use so that its operation can draw back the absorbentmaterial 200 for removal from the vagina. In alternate embodiments, thetether 405 can be coiled inside the cavity 408 and is deployed as theabsorbent material 200 is deployed. The proximal end of the tether 405can be such that it cannot pass through the distal end of the housing402. In this way, once the housing 402 is removed, the remaining tetheris uncoiled and can be extended to its full length. Once the housing 402clears the vagina the tether can be separated to allow the housing to beremoved completely.

As noted above, the absorbent material 200 can remain in the vaginalcanal V for a set amount of time, from minutes or hours, to adequatelyabsorb the maternal vaginal microbiota. During the wait time, thedeployment/retrieval device 400 can be prepared for removal of theabsorbent material 200 or a separate device can be used. Here, thedeployment/retrieval element 404 can either be removed, or used tofacilitate the “threading” of the tether 405 back into the device 400through the cavity 408. The cavity 408 itself can be resterilized and/orcoated with substances 416 that can maintain/support the maternalvaginal microbiota.

At the appropriate time for removal, the deployment/retrieval device 400can be reintroduced into the vaginal canal V. The tether 405 can bethreaded back through the deployment/retrieval device 400 and theabsorbent material 200 can begin to be retrieved into the cavity 408,which is now acting like a sterile container. FIG. 10D illustrates thepartial capture of the absorbent material 200 into the cavity 408. FIG.10E illustrates an embodiment of the final retrieval step in which theabsorbent material 200 can be stored in a sealed container, which is, inthis embodiment, the cavity 408, until use.

In another embodiment, the absorbent material 200 can be wrapped insterile packaging and the cavity 408 may not sterile. The seal 412A canbe a distal end of the sterile packaging designed to befrangible/tearable/breakable upon pressure from the deployment/retrievalelement 404 as it displaces the absorbent material 200 into the vaginalcanal V. The absorbent material 200 can have a reattachable interface407 with matching interface on the deployment/retrieval element 404. Therear seal 412B acts as a barrier between the interfaces 407 duringdeployment so the absorbent material 200 can be deployed without havingto disengage the interfaces. Once the sterile packaging is removed fromthe housing 102, the reattachable interfaces 407 can now engage when theabsorbent material 200 is being retrieved. See, for example, FIGS. 10Fand 10G.

The container 400 can, in certain embodiments, just be a sterile vessel.In other embodiments, it can have substances that can maintain/supportthe maternal vaginal microbiota. The substances can, in certainembodiments, control pH, provided growth media, inhibit “bad”microbiota, and/or stabilize the maternal vaginal microbiota for longterm storage, e.g. freeze drying.

A kit can be available with all of the elements necessary to collect,retrieve, dispense to the infant, and store maternal vaginal fluids,and/or the maternal vaginal microbiota This kit can include one or moreof the element above, combined together and packaged together for easeof use.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

The invention provides the following Examples in which newborns born byC-section were exposed to a gauze that had been previously introduced inthe maternal vagina for one hour prior to the surgical procedure. Thegauze was obtained from healthy mothers with acidic, Lactobacillusdominant, Group B Streptococcus-negative vaginas. Newborns wereimmediately exposed to the gauze at birth, passing the gauze firstthrough the mouth, then the rest of the face and body. Swabs from oral,skin, and anal regions were taken from the baby and the mother (fromwhom vaginal swabs were also obtained). Sampling was done at 6 timepoints during the first month of life, starting at the day of birth.Bacterial DNA was extracted and the V4 region of the 16S rRNA gene wassequenced using an Illumina sequencing instrument. The bacterialmicrobiota from the multiple body sites was analyzed using QIIME.Details of the materials and study designs, as well as the results, areprovided as follows.

Example 1: Transferring Maternal Vaginal Microbiome to Infants UnderC-Section Deliveries Materials and Methods

Study Design and Enrollment Criteria

The study protocol was approved by the Institutional Review Board of theUniversity of Puerto Rico Recinto de Ciencias Medicas and Rio Piedrascampus. Mothers were consented during their 3rd trimester control OBGYNcontrol visit. There were three groups of mothers, by delivery andnewborn exposure, and they included vaginal, C-section and C-sectionwith exposure to maternal vaginal contents. Inclusion criteria includedhealthy mothers over 21 years of age, with uncomplicated pregnancies.C-sections were all scheduled, mostly due to previous C-section ormaternal choice. For the C-section with exposure to maternal vaginalfluids group, mothers had to have vaginal pH<4 at the time of birth (asmeasured with a vaginal swab on pH paper). 18 mothers were recruited forthis preliminary study, 7 of which gave birth vaginally and 11 byscheduled C-section, of which 4 newborns were exposed at birth to theirmother's vaginal contents (Table 2).

TABLE 2 Mode and location of delivery of the 20 mothers of the studyPerinatal Family # Mode of delivery Birth Location GBS antibiotics 1Vaginal Home − − 15 Vaginal Hospital

16 Vaginal Hospital − − 19 Vaginal Hospital − − 20 Vaginal Hospital − −21 Vaginal Home − − 22 Vaginal Hospital − − 2 Cesarean Hospital −

3 Cesarean Hospital −

4 Cesarean Hospital −

5 Cesarean Hospital −

6 Cesarean Hospital −

8 Cesarean Hospital −

9 Cesarean Hospital −

10 Cesarean Hospital

11 Cesarean Hospital

13 Cesarean + Exposure Hospital −

14 Cesarean + Exposure Hospital −

17 Cesarean + Exposure Hospital −

18 Cesarean + Exposure Hospital −

Procedure of Gauze Exposure in C-Section Babies

Mothers were sampled before the C-section prophylactic antibioticsadministration. A pair of vaginal swabs were taken, one to measure fluidpH using pH strip paper (Lab Mikro Hydrion™ pH Test Paper, Fisher13-640-508). By the time the mother is administered the preventiveantibiotics (1 g Penicillin-family antibiotics), a medium sterile padgauze (J&J, 7.6×76 cm) folded like a fan and then in half, was wet withsterile saline solution and introduced in the maternal birth canal inthe hour prior to the C-section, at the time antibiotics wereadministered. The surgeon extracted the gauze right before theprocedure, and it was used to swab the infant just after birth (FIG. 3).

Sample Collection

Pre-labeled swabs—in duplicate—were taken from 5 body sites of motherand baby (oral mucosa, forehead, right volar arm, right foot, and anal)plus two additional from the mother (right aureole, vagina; Table 3).

TABLE 3 Number of samples from mothers and infants Cesarean + C-sectionExposure Vaginal Body site N swabs Mother Infant Mother Infant MotherInfant Total Vaginal 78 32 — 15 — 31 — 78 Aureole 98 39 — 17 — 42 — 98skin 548 168 104 60 30 93 93 548 anal 226 33 34 14 50 62 33 226 oral 18542 44 15 35 30 19 185 Total 1057 282 182 106 115 227 145 1057

Samples were collected from the mother before birth, and after birth ateach timepoint of the mother-baby pair sampling, namely at ˜day 1, 3, 7,and weekly thereafter to the first month (Table 2). Vials weremaintained cold and frozen at ultralow temperature (−70 Celsius orbelow) within the following 2 hours of collection.

At each time point at which samples were collected information surveywas applied, and information about mother and baby health, dietarychanges and medications was collected. 16S rRNA sequencing and analyses

Bacterial DNA was extracted from the 1057 swabs, and the V4 region ofthe 16S rRNA was amplified and sequenced using Illumina HiSeq aspreviously described (43). Alpha and beta diversity were estimated usingQiime (33). Linear Discriminant Analysis Effect Size (LEfSe) (44) withdefault parameters was used to determine taxa that was overrepresentedin each baby group in relation to another.

Results

Samples from 18 infants and their mothers (Table 1) were analyzed,including 7 born vaginally and 11 delivered by scheduled cesarean, ofwhich 4 were exposed to the maternal vaginal fluids at birth, using asterile gauze (Table 1). After transferring the maternal vaginalmicrobiota to the newborn, the infant microbiota of the exposed groupwas compared with those from infants born by C-section without exposure,during the first month of life. Briefly, the procedure involvedincubating a gauze in the maternal vagina, for the hour preceding theC-section, in mothers that complied with inclusion criteria (scheduledC-section, negative results for GBS, HIV, Chlamydia; vaginal pH<4.5 asmeasured with a vaginal swab sample on a pH paper strip). Within thefirst few minutes after birth (1-3 min) of these cesarean-deliveredinfants, the newborns were exposed to their mother's vaginal contentsswabbing the newborn body, mouth first, then face and rest of the body(FIG. 1 ). Of the 14 mothers whose infants were not exposed to thegauze, 3 were GBS positive, 2 delivering by C-section and 1 deliveringvaginally. All mothers who underwent Cesarean section received perinatalantibiotics, and the one GBS positive mother who delivered vaginally.

A total of 1072 swabs from multiple body sites were obtained from the 18babies and mothers, during the first month of life (at 1, 3, 7, 14, 21and 30 days after the birth). Bacterial communities were characterizedby Illumina sequencing of the V4 region of 16S rRNA gene. Samples thathad >1,000 sequences (n=1016) were further analyzed. A total of6,515,724 sequences were obtained (mean 6,(32)3±4,593, median 5,360sequences), and assigned to taxa using open reference operationaltaxonomic unit (OTU) picking using Qiime (33).

Body site differentiation of colonized sites in newborns occurred in asfew as 3 days for skin and mouth, but not for in the whole first monthfor anal communities (FIG. 2 ). Regardless of exposure or birth mode,oral and anal—but not skin—sites showed the highest bacterial diversityat birth, and alpha diversity decreased soon after birth (by day 3) inoral and anal sites, and remained relative stable during the first monthof life (FIGS. 3 & 4 ), when communities seem to converge (FIG. 5 ).Despite convergence in whole community structure, major differencessegregate apart the microbiota of infants, by mode of delivery andexposure.

The major bacterial markers of delivery present in vaginally born andnot in unexposed Cesarean-delivered infants were i) Lactobacillus,present in maternal vagina and in all infant sites at birth, showing areduction concomitantly with the reduction in site alpha diversity,during the first 3 days after birth (FIGS. 2 & 6 ); ii) Bacteroides,Clostridium and Bifidobacterium in anal swabs; Streptococcus andStaphylococcus in skin and mouth; S24-7 and Stenotrophomonas in skin andoral Veillonella and Gemellaceae, which bloomed during the first week ofthe baby life and remained relatively stable throughout the first month,only in vaginally born infants (FIG. 2 ). In contrast, unexposedC-section born babies had overrepresented anal Veillonella (FIG. 2 ).

To identify more specifically bacterial species present in vaginallyborn and not in unexposed Cesarean-delivered infants, DNA was extractedand the V4 region of 16S rRNA gene was sequenced in Illumina. Sequenceswere assigned to taxa using BLASTN (web-based blastn site, NCBI) usingOTU picking algorithm, 97% identity to the Greengenes database (v13_8),in QIIME (Caporaso et al., 2010, Nat. Methods, 7(5): 335-336). Tofurther confirm taxonomies, the 16S rRNA sequences of each OTU were usedin BLASTN (web-based blastn suite, NCBI) using 97% identity to theGenbank 16S rRNA sequence database (May 2016). The results are shown inTable 4.

TABLE 4 Species-Level Ideintification of Bacteria DifferentiallyObserved in Infants by Exposure at Birth BLASTN Taxonomic info Genusspecies Identity (%) p_Proteobacteria; c_Gammaproteobacteria;Acinetobacter 100 o_Pseudomonadales; f_Moraxellaceae; baumanniig_Acinetobacter; s_(—) p_Proteobacteria; c_Gammaproteobacteria;Acinetobacter 100 o_Pseudomonadales; f_Moraxellaceae; gernerig_Acinetobacter; s_(—) p_Proteobacteria; c_Gammaproteobacteria;Acinetobacter 98.42 o_Pseudomonadales; f_Moraxellaceae; johnsoniig_Acinetobacter; s_johnsonii p_Proteobacteria; c_Gammaproteobacteria;Acinetobacter 99.6 o_Pseudomonadales; f_Moraxellaceae; radioresistensg_Acinetobacter; s_(—) p_Proteobacteria; c_Gammaproteobacteria;Acinetobacter 98.42 o_Pseudomonadales; f_Moraxellaceae; schindlerig_Acinetobacter; s_(—) p_Proteobacteria; c_Gammaproteobacteria;Acinetobacter 99.6 o_Pseudomonadales; f_Moraxellaceae; seifertiig_Acinetobacter; s_rhizosphaerae p_Proteobacteria;c_Gammaproteobacteria; Acinetobacter 100 o_Pseudomonadales;f_Moraxellaceae; variabilis g_Acinetobacter; s_(—) p_Actinobacteria;c_Actinobacteria; Actinomyces 100 o_Actinomycetales; f_Actinomycetaceae;europaeus g_Actinomyces; s_europaeus p_Actinobacteria; c_Actinobacteria;Actinomyces neuii 100 o_Actinomycetales; f_Actinomycetaceae;g_Actinomyces; s_(—) p_Firmicutes; c_Bacilli; o_Lactobacillales;Aerococcus 99.21 f_Aerococcaceae; g_Aerococcus; s_(—) christenseniip_Actinobacteria; c_Actinobacteria; Alloscardovia 100o_Bifidobacteriales; f_Bifidobacteriaceae; g_; s_(—) omnicolensp_Firmicutes; c_Clostridia; o_Clostridiales; Anaerococcus 100f_[Tissierellaceae]; g_Anaerococcus; s_(—) lactolyticus p_Firmicutes;c_Clostridia; o_Clostridiales; Anaerococcus 100 f_[Tissierellaceae];g_Anaerococcus; s_(—) murdochii p_Firmicutes; c_Clostridia;o_Clostridiales; Anaerococcus 100 f_[Tissierellaceae]; g_Anaerococcus;s_(—) obesiensis p_Firmicutes; c_Clostridia; o_Clostridiales;Anaerococcus 100 f_[Tissierellaceae]; g_Anaerococcus; s_(—) octaviusp_Firmicutes; c_Clostridia; o_Clostridiales; Anaerococcus 100f_[Tissierellaceae]; g_Anaerococcus; s_(—) prevotii p_Firmicutes;c_Clostridia; o_Clostridiales; Anaerococcus 100 f_[Tissierellaceae];g_Anaerococcus; s_(—) provenciensis p_Firmicutes; c_Clostridia;o_Clostridiales; Anaerococcus 100 f_[Tissierellaceae]; g_Anaerococcus;s_(—) vaginalis p_Firmicutes; c_Clostridia; o_Clostridiales;Anaerostipes hadrus 99.21 f_Lachnospiraceae; g_; s_(—) p_Firmicutes;c_Bacilli; o_Bacillales; Anoxybacillus 100 f_Bacillaceae;g_Anoxybacillus; s_kestanbolensis flavithermus p_Proteobacteria;c_Alphaproteobacteria; Asticcacaulis 97.23 o_Caulobacterales;f_Caulobacteraceae; excentricus g_Asticcacaulis; s_(—) p_Actinobacteria;c_Coriobacteriia; Atopobium deltae 99.61 o_Coriobacteriales;f_Coriobacteriaceae; g_Atopobium; s_(—) p_Actinobacteria;c_Coriobacteriia; Atopobium vaginae 100 o_Coriobacteriales;f_Coriobacteriaceae; g_; s_(—) p_Firmicutes; c_Bacilli; o_Bacillales;Bacillus vireti 100 f_Bacillaceae; g_Bacillus; s_(—) p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; Bacteroides 98.42 f_Bacteroidaceae;g_Bacteroides; s_(—) finegoldii p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; Bacteroides vulgatus 100 f_Bacteroidaceae;g_Bacteroides; s_(—) p_Actinobacteria; c_Actinobacteria; Bifidobacterium100 o_Bifidobacteriales; f_Bifidobacteriaceae; breve g_Bifidobacterium;s_(—) p_Actinobacteria; c_Actinobacteria; Bifidobacterium 98.81o_Bifidobacteriales; f_Bifidobacteriaceae; pseudolongumg_Bifidobacterium; s_(—) p_Proteobacteria; c_Alphaproteobacteria;Blastomonas 100 o_Sphingomonadales; f_Sphingomonadaceae; g_; s_(—)natatoria p_Proteobacteria; c_Alphaproteobacteria; Bradyrhizobium 100o_Rhizobiales; f_Bradyrhizobiaceae; g_; s_(—) lupini p_Actinobacteria;c_Actinobacteria; Brevibacterium 99.21 o_Actinomycetales;f_Brevibacteriaceae; paucivorans g_Brevibacterium; s_paucivoransp_Proteobacteria; c_Epsilonproteobacteria; Campylobacter coli 100o_Campylobacterales; f_Campylobacteraceae; g_Campylobacter; s_(—)p_Proteobacteria; c_Epsilonproteobacteria; Campylobacter 100o_Campylobacterales; f_Campylobacteraceae; hominis g_Campylobacter;s_(—) p_Proteobacteria; c_Epsilonproteobacteria; Campylobacter 100o_Campylobacterales; f_Campylobacteraceae; ureolyticus g_Campylobacter;s_(—) p_Actinobacteria; c_Actinobacteria; Clavibacter 98.42o_Actinomycetales; f_Microbacteriaceae michiganensis p_Bacteroidetes;c_Flavobacteriia; Cloacibacterium 99.21 o_Flavobacteriales;f_[Weeksellaceae]; rupense g_Cloacibacterium; s_(—) p_Firmicutes;c_Clostridia; o_Clostridiales; Clostridium 100 f_Lachnospiraceae; g_;s_(—) clostridioforme p_Firmicutes; c_Clostridia; o_Clostridiales;Clostridium N/A f_Clostridiaceae; g_Clostridium; s_perfringensperfringens p_Proteobacteria; c_Betaproteobacteria; Comamonas 99.21o_Burkholderiales; f_Comamonadaceae; g_; s_(—) serinivoransp_Proteobacteria; c_Betaproteobacteria; Comamonas 100 o_Burkholderiales;f_Comamonadaceae; testosteroni g_Comamonas; s_(—) p_Actinobacteria;c_Actinobacteria; Corynebacterium 98.43 o_Actinomycetales;f_Corynebacteriaceae; amycolatum g_Corynebacterium; s_(—)p_Actinobacteria; c_Actinobacteria; Corynebacterium 100o_Actinomycetales; f_Corynebacteriaceae; appendicis g_Corynebacterium;s_(—) p_Actinobacteria; c_Actinobacteria; Corynebacterium 99.21o_Actinomycetales; f_Corynebacteriaceae; argentoratenseg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae; aurimucosumg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 99.61 o_Actinomycetales; f_Corynebacteriaceae; canisg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 97.24 o_Actinomycetales; f_Corynebacteriaceae; caseig_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae; coyleaeg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae; freneyig_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 98.82 o_Actinomycetales; f_Corynebacteriaceae; imitansg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae; jeikeiumg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae;kroppenstedtii g_Corynebacterium; s_kroppenstedtii p_Actinobacteria;c_Actinobacteria; Corynebacterium 100 o_Actinomycetales;f_Corynebacteriaceae; lactis g_Corynebacterium; s_(—) p_Actinobacteria;c_Actinobacteria; Corynebacterium 99.21 o_Actinomycetales;f_Corynebacteriaceae; matruchotii g_Corynebacterium; s_(—)p_Actinobacteria; c_Actinobacteria; Corynebacterium 98.31o_Actinomycetales; f_Corynebacteriaceae; mycetoides g_Corynebacterium;s_(—) p_Actinobacteria; c_Actinobacteria; Corynebacterium 99.61o_Actinomycetales; f_Corynebacteriaceae; mycetoides g_Corynebacterium;s_(—) p_Actinobacteria; c_Actinobacteria; Corynebacterium 100o_Actinomycetales; f_Corynebacteriaceae; pilbarense g_Corynebacterium;s_(—) p_Actinobacteria; c_Actinobacteria; Corynebacterium 100o_Actinomycetales; f_Corynebacteriaceae; pyruviciproducensg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae;spheniscorum g_Corynebacterium; s_(—) p_Actinobacteria;c_Actinobacteria; Corynebacterium 100 o_Actinomycetales;f_Corynebacteriaceae; striatum g_Corynebacterium; s_(—)p_Actinobacteria; c_Actinobacteria; Corynebacterium 99.21o_Actinomycetales; f_Corynebacteriaceae; terpenotabidumg_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae; thomsseniig_Corynebacterium; s_(—) p_Actinobacteria; c_Actinobacteria;Corynebacterium 100 o_Actinomycetales; f_Corynebacteriaceae;tuberculostearicum g_Corynebacterium; s_(—) p_Actinobacteria;c_Actinobacteria; Corynebacterium 99.61 o_Actinomycetales;f_Corynebacteriaceae; tuscaniense g_Corynebacterium; s_(—)p_Actinobacteria; c_Actinobacteria; Dermabacter 99.6 o_Actinomycetales;f_Dermabacteraceae; hominis g_Dermabacter; s_(—) p_Actinobacteria;c_Actinobacteria; Devriesea 97.63 o_Actinomycetales; f_Dermabacteraceaeagamarum p_Firmicutes; c_Clostridia; o_Clostridiales; Dialister 100f_Veillonellaceae; g_Dialister; s_(—) micraerophilus p_Firmicutes;c_Clostridia; o_Clostridiales; Dialister 100 f_Veillonellaceae;g_Dialister; s_(—) propionicifaciens p_Firmicutes; c_Clostridia;o_Clostridiales; Dialister 97.62 f_Veillonellaceae; g_Dialister; s_(—)succinatiphilus p_Firmicutes; c_Clostridia; o_Clostridiales; Dorealongicatena 99.61 f_Lachnospiraceae; g_[Ruminococcus]; s_gnavusp_Firmicutes; c_Bacilli; o_Lactobacillales; Enterococcus hirae 100f_Enterococcaceae; g_Enterococcus; s_(—) p_Firmicutes; c_Bacilli;o_Lactobacillales; Eremococcus 100 f_Aerococcaceae; g_Facklamia; s_(—)coleocola p_Firmicutes; c_Erysipelotrichi; Erysipelatoclostridium 100o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s_(—) ramosump_Proteobacteria; c_Gammaproteobacteria; Escherichia 100o_Enterobacteriales; f_Enterobacteriaceae; g_; s_(—) marmotaep_Firmicutes; c_Clostridia; o_Clostridiales; Ezakiella peruensis 100f_[Tissierellaceae]; g_1-68; s_(—) p_Firmicutes; c_Bacilli;o_Lactobacillales; Facklamia hominis 99.21 f_Aerococcaceae; g_Facklamia;s_(—) p_Firmicutes; c_Bacilli; o_Lactobacillales; Facklamia ignava 100f_Aerococcaceae; g_Facklamia; s_(—) p_Firmicutes; c_Clostridia;o_Clostridiales; Fastidiosipila 99.21 f_Clostridiaceae; g_Clostridium;s_(—) sanguinis p_Firmicutes; c_Clostridia; o_Clostridiales; Fenollaria100 f_[Tissierellaceae]; g_WAL_1855D; s_(—) massiliensis p_Firmicutes;c_Clostridia; o_Clostridiales; Finegoldia magna 100 f_[Tissierellaceae];g_Finegoldia; s_(—) p_Fusobacteria; c_Fusobacteriia; Fusobacterium 100o_Fusobacteriales; f_Fusobacteriaceae; equinum g_Fusobacterium; s_(—)p_Fusobacteria; c_Fusobacteriia; Fusobacterium 100 o_Fusobacteriales;f_Fusobacteriaceae; nucleatum g_Fusobacterium; s_(—) p_Fusobacteria;c_Fusobacteriia; Fusobacterium 97.22 o_Fusobacteriales;f_Fusobacteriaceae; periodonticum g_Fusobacterium; s_(—) p_Fusobacteria;c_Fusobacteriia; Fusobacterium 100 o_Fusobacteriales;f_Fusobacteriaceae; simiae g_Fusobacterium; s_(—) p_Actinobacteria;c_Actinobacteria; Gardnerella 100 o_Bifidobacteriales;f_Bifidobacteriaceae vaginalis p_Firmicutes; c_Bacilli; o_Gemellales;Gemella 99.6 f_Gemellaceae; g_Gemella; s_(—) asaccharolyticap_Firmicutes; c_Bacilli; o_Gemellales; Gemella taiwanensis 100f_Gemellaceae; g_; s_(—) p_Proteobacteria; c_Gammaproteobacteria;Haemophilus 100 o_Pasteurellales; f_Pasteurellaceae; pittmaniaeg_Aggregatibacter; s_(—) p_Firmicutes; c_Clostridia; o_Clostridiales;Helcococcus 100 f_[Tissierellaceae]; g_Helcococcus; s_(—) sueciensisp_Proteobacteria; c_Betaproteobacteria; Herbaspirillum 100o_Burkholderiales; f_Oxalobacteraceae chlorophenolicum p_Synergistetes;c_Synergistia; o_Synergistales; Jonquetella anthropi 100f_Dethiosulfovibrionaceae; g_Jonquetella; s_anthropi p_Actinobacteria;c_Actinobacteria; Kocuria flava strain 98.42 o_Actinomycetales;f_Micrococcaceae; HO-9041 g_Microbispora; s_rosea p_Actinobacteria;c_Actinobacteria; Kocuria kristinae 100 o_Actinomycetales;f_Micrococcaceae; g_; s_(—) strain DSM 20032 p_Actinobacteria;c_Actinobacteria; Kytococcus 99.53 o_Actinomycetales;f_Intrasporangiaceae schroeteri strain Muenster 2000 p_Firmicutes;c_Bacilli; o_Lactobacillales; Lactobacillus 100 f_Lactobacillaceae;g_Lactobacillus; s_(—) coleohominis p_Firmicutes; c_Bacilli;o_Lactobacillales; Lactobacillus 100 f_Lactobacillaceae;g_Lactobacillus; s_(—) crispatus p_Firmicutes; c_Bacilli;o_Lactobacillales; Lactobacillus 100 f_Lactobacillaceae;g_Lactobacillus; s_(—) hominis p_Firmicutes; c_Bacilli;o_Lactobacillales; Lactobacillus iners 100 f_Lactobacillaceae;g_Lactobacillus; s_iners p_Firmicutes; c_Bacilli; o_Lactobacillales;Lactobacillus 100 f_Lactobacillaceae; g_Lactobacillus; s_(—)intestinalis p_Firmicutes; c_Bacilli; o_Lactobacillales; Lactobacillus100 f_Lactobacillaceae; g_Lactobacillus; s_(—) jensenii p_Firmicutes;c_Bacilli; o_Lactobacillales; Lactobacillus 98.81 f_Lactobacillaceae;g_Lactobacillus; s_(—) psittaci p_Firmicutes; c_Bacilli;o_Lactobacillales; Lactobacillus reuteri 99.6 f_Lactobacillaceae;g_Lactobacillus; s_(—) p_Firmicutes; c_Bacilli; o_Lactobacillales;Lactobacillus 97.23 f_Lactobacillaceae; g_Lactobacillus; s_(—) rodentiump_Firmicutes; c_Bacilli; o_Lactobacillales; Lactococcus lactis 99.6f_Streptococcaceae; g_Lactococcus; s_(—) p_Firmicutes; c_Clostridia;o_Clostridiales; Levyella massiliensis 100 f_[Tissierellaceae]; g_ph2;s_(—) p_Proteobacteria; c_Betaproteobacteria; Methylobacillus 100o_Methylophilales; f_Methylophilaceae flagellatus p_Proteobacteria;c_Alphaproteobacteria; Methylobacterium 100 o_Rhizobiales;f_Methylobacteriaceae; aerolatum g_Methylobacterium p_Proteobacteria;c_Alphaproteobacteria; Methylobacterium 99.6 o_Rhizobiales;f_Methylobacteriaceae; phyllostachyos g_Methylobacterium; s_(—)p_Actinobacteria; c_Actinobacteria; Micrococcus 100 o_Actinomycetales;f_Micrococcaceae; aloeverae strain AE- g_Micrococcus; s_(—) 6p_Actinobacteria; c_Actinobacteria; Mobiluncus curtisii 100o_Actinomycetales; f_Actinomycetaceae; g_Mobiluncus; s_(—)p_Tenericutes; c_Mollicutes; Mycoplasma 100 o_Mycoplasmatales;f_Mycoplasmataceae; hominis g_Mycoplasma; s_(—) p_Firmicutes;c_Clostridia; o_Clostridiales; Negativicoccus 100 f_Veillonellaceaesuccinicivorans p_Actinobacteria; c_Actinobacteria; Oceanitalea 98.42o_Actinomycetales; f_Bogoriellaceae; nanhaiensis g_Georgenia; s_(—)p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; Parabacteroides 98.42f_Porphyromonadaceae; g_Parabacteroides; s_(—) faecis p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; Parabacteroides 100f_Porphyromonadaceae; g_Parabacteroides; s_(—) merdae p_Proteobacteria;c_Alphaproteobacteria; Paracoccus 98.81 o_Rhodobacterales;f_Rhodobacteraceae; g_; s_(—) communis p_Firmicutes; c_Clostridia;o_Clostridiales; Parvimonas micra 97.62 f_[Tissierellaceae];g_Parvimonas; s_(—) p_Proteobacteria; c_Betaproteobacteria; Pelistegaindica 99.61 o_Burkholderiales; f_Alcaligenaceae; g_; s_(—)p_Firmicutes; c_Clostridia; o_Clostridiales; Peptococcus niger 98.81f_Peptococcaceae; g_Peptococcus; s_(—) p_Firmicutes; c_Clostridia;o_Clostridiales; Peptoniphilus coxii 100 f_[Tissierellaceae];g_Peptoniphilus; s_(—) p_Firmicutes; c_Clostridia; o_Clostridiales;Peptoniphilus 100 f_[Tissierellaceae]; g_Peptoniphilus; s_(—) duerdeniip_Firmicutes; c_Clostridia; o_Clostridiales; Peptoniphilus 100f_[Tissierellaceae]; g_Peptoniphilus; s_(—) grossensis p_Firmicutes;c_Clostridia; o_Clostridiales; Peptoniphilus 99.6 f_[Tissierellaceae];g_Peptoniphilus; s_(—) koenoeneniae p_Firmicutes; c_Clostridia;o_Clostridiales; Peptoniphilus 100 f_[Tissierellaceae]; g_Peptoniphilus;s_(—) lacrimalis p_Firmicutes; c_Clostridia; o_Clostridiales;Peptoniphilus obesi 98.02 f_[Tissierellaceae]; g_Peptoniphilus; s_(—)strain ph1 p_Firmicutes; c_Clostridia; o_Clostridiales; Peptoniphilus97.12 f_[Tissierellaceae]; g_Peptoniphilus; s_(—) senegalensisp_Firmicutes; c_Clostridia; o_Clostridiales; Peptoniphilus 100f_[Tissierellaceae]; g_Peptoniphilus; s_(—) tyrrelliae p_Firmicutes;c_Clostridia; o_Clostridiales; Peptostreptococcus 100f_Peptostreptococcaceae; g_Peptostreptococcus; anaerobius s_anaerobiusp_Proteobacteria; c_Betaproteobacteria; Polaromonas sp 99.21o_Burkholderiales; f_Comamonadaceae p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; Porphyromonas 99.21 f_Porphyromonadaceae;g_Porphyromonas; s_(—) bennonis p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; Porphyromonas 100 f_Porphyromonadaceae;g_Porphyromonas; s_(—) somerae p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; Porphyromonas 99.6 f_Porphyromonadaceae;g_Porphyromonas; s_(—) uenonis p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; Prevotella amnii 99.6 f_Prevotellaceae; g_Prevotella;s_(—) p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; Prevotellabergensis 100 f_Prevotellaceae; g_Prevotella; s_(—) p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; Prevotella bivia 100 f_Prevotellaceae;g_Prevotella; s_(—) p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;Prevotella buccalis 99.6 f_Prevotellaceae; g_Prevotella; s_(—)p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; Prevotella copri 100f_Prevotellaceae; g_Prevotella; s_copri p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; Prevotella corporis 100 f_Prevotellaceae; g_Prevotella;s_(—) p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; Prevotelladisiens 100 f_Prevotellaceae; g_Prevotella; s_(—) p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; Prevotella 100 f_Prevotellaceae;g_Prevotella; s_(—) timonensis p_Actinobacteria; c_Actinobacteria;Pseudoclavibacter 99.21 o_Actinomycetales; f_Microbacteriaceae; bifidastrain IAM g_Pseudoclavibacter; s_bifida 14848 p_Proteobacteria;c_Gammaproteobacteria; Pseudomonas 100 o_Pseudomonadales;f_Pseudomonadaceae; g_; s_(—) brenneri p_Proteobacteria;c_Gammaproteobacteria; Pseudomonas 99.6 o_Pseudomonadales;f_Pseudomonadaceae; helmanticensis g_Pseudomonas; s_(—)p_Proteobacteria; c_Gammaproteobacteria; Pseudomonas lini 100o_Pseudomonadales; f_Pseudomonadaceae; g_Pseudomonas; s_(—)p_Proteobacteria; c_Gammaproteobacteria; Pseudomonas 99.21o_Pseudomonadales; f_Pseudomonadaceae; syringae g_Pseudomonas; s_(—)p_Actinobacteria; c_Actinobacteria; Rothia amarae 97.23o_Actinomycetales; f_Micrococcaceae; g_; s_(—) strain J18p_Actinobacteria; c_Actinobacteria; Rothia mucilaginosa 98.82o_Actinomycetales; f_Micrococcaceae; g_Rothia; s_mucilaginosap_Actinobacteria; c_Rubrobacteria; Rubrobacter 98.42 o_Rubrobacterales;f_Rubrobacteraceae; calidifluminis g_Rubrobacter; s_(—) p_Firmicutes;c_Clostridia; o_Clostridiales; Ruminococcus 98.43 f_Ruminococcaceae;g_Ruminococcus; s_(—) bromii p_Firmicutes; c_Clostridia;o_Clostridiales; Ruminococcus 100 f_Lachnospiraceae; g_[Ruminococcus];s_gnavus gnavus p_Fusobacteria; c_Fusobacteriia; Sneathia 100o_Fusobacteriales; f_Leptotrichiaceae; sanguinegens g_Sneathia; s_(—)p_Proteobacteria; c_Alphaproteobacteria; Sphingobium 100o_Sphingomonadales; f_Sphingomonadaceae; yanoikuyae g_Sphingobium; s_(—)p_Proteobacteria; c_Alphaproteobacteria; Sphingopyxis 99.6o_Sphingomonadales; f_Sphingomonadaceae; macrogoltabida g_Sphingopyxis;s_(—) p_Firmicutes; c_Bacilli; o_Bacillales; Staphylococcus 100f_Planococcaceae; g_; s_(—) carnosus p_Firmicutes; c_Bacilli;o_Bacillales; Staphylococcus 97.23 f_Staphylococcaceae; g_Staphylococcuschromogenes p_Firmicutes; c_Bacilli; o_Bacillales; Staphylococcus 100f_Staphylococcaceae; g_Staphylococcus; s_(—) petrasii p_Firmicutes;c_Bacilli; o_Bacillales; Staphylococcus 97.23 f_Staphylococcaceae;g_Staphylococcus pseudintermedius p_Firmicutes; c_Bacilli; o_BacillalesStaphylococcus 100 saprophyticus p_Proteobacteria;c_Gammaproteobacteria; Stenotrophomonas 98.81 o_Xanthomonadales ;f_Xanthomonadaceae; maltophilia g_Stenotrophomonas; s_(—) p_Firmicutes;c_Bacilli; o_Lactobacillales; Streptococcus N/A f_Streptococcaceae;g_Streptococcus; agalactiae s_agalactiae p_Firmicutes; c_Bacilli;o_Lactobacillales; Streptococcus 100 f_Streptococcaceae;g_Streptococcus; s_(—) agalactiae p_Firmicutes; c_Bacilli;o_Lactobacillales; Streptococcus 100 f_Streptococcaceae;g_Streptococcus; anginosus s_anginosus p_Firmicutes; c_Bacilli;o_Lactobacillales; Streptococcus 97.62 f_Streptococcaceae;g_Streptococcus; constellatus s_anginosus p_Firmicutes; c_Bacilli;o_Lactobacillales; Streptococcus 100 f_Streptococcaceae;g_Streptococcus; s_(—) dentisani p_Firmicutes; c_Bacilli;o_Lactobacillales; Streptococcus 99.6 f_Streptococcaceae;g_Streptococcus; s_(—) lactarius p_Firmicutes; c_Bacilli;o_Lactobacillales; Streptococcus 99.21 f_Streptococcaceae;g_Streptococcus; s_(—) thermophilus p_Proteobacteria;c_Betaproteobacteria; Sutterella 99.6 o_Burkholderiales;f_Alcaligenaceae; stercoricanis g_Sutterella; s_(—) p_Tenericutes;c_Mollicutes; Ureaplasma 100 o_Mycoplasmatales; f_Mycoplasmataceae;urealyticum g_Ureaplasma; s_(—) p_Actinobacteria; c_Actinobacteria;Varibaculum 100 o_Actinomycetales; f_Actinomycetaceae; cambrienseg_Varibaculum; s_(—) p_Firmicutes; c_Clostridia; o_Clostridiales;Veillonella dispar 100 f_Veillonellaceae; g_Veillonella; s_disparp_Firmicutes; c_Clostridia; o_Clostridiales; Veillonella ratti 99.6f_Veillonellaceae; g_Veillonella; s_(—) p_Firmicutes; c_Clostridia;o_Clostridiales; f_[Tissierellaceae]; g_1-68; s_(—) p_Firmicutes;c_Clostridia; o_Clostridiales; f_Veillonellaceae; g_Megasphaera; s_(—)p_Fusobacteria; c_Fusobacteriia; o_Fusobacteriales; f_Leptotrichiaceae;g_Leptotrichia; s_(—) p_Proteobacteria; c_Alphaproteobacteria;o_Sphingomonadales; f_Sphingomonadaceae; g_Novosphingobium; s_(—)p_Proteobacteria; c_Betaproteobacteria; o_Neisseriales; f_Neisseriaceae;g_; s_(—) p_Proteobacteria; c_Gammaproteobacteria; o_Xanthomonadales;f_Xanthomonadaceae; g_Stenotrophomonas; s_(—)

Example 2: Restoration by Exposure to Vaginal Fluids in C-SectionDelivered Infants

Since Cesarean-delivered infants were exposed to vaginal fluids throughthe use of sterile gauzes, the similarity of the microbiota of thegauzes to samples obtained from maternal body sites at day 1 wasdetermined. The gauzes contained vaginal bacteria such as Lactobacillus,Prevotella, Garnerella, and in less proportion by Atopobium, Sneathia,Fusobacterium, and Ureoplasma, and were more similar to the microbiotaof maternal vagina, than to that in other body sites (ANOVA p<0.01).

Vaginal gauze exposure lead to an infant microbiota closer to thattypical of vaginal than C-section delivery, with partially restoredvaginal markers that were lacking in C-section infants (FIG. 2 , FIG. 6, Table 5). Major restored bacteria included Lactobacillus in all infantsites and the early bloom of gut Bacteroides, oral Gemella, and skinBacteroidales S24-7 and Stenotrophomonas. However, these gauze-exposedC-section babies also showed some markers of C-section delivery, such ashigh anal Streptococcus (FIG. 2 , Table 5). Random Forest predictionsindicated that mode of delivery could be predicted using oral or skinmicrobes. C section birthing was more accurately predicted than vaginaldelivery, and mode of delivery of babies born by C-section with gauzeexposure could not be predicted, indicating an intermediate composition.

TABLE 5 Bacteria differentially observed in infants by exposure at birthBacterial genus Delivery mode Vaginal Cesarean Vaginal exposure at

− birth Infant body site Lactobacillus All

− Bacteroides Anal

− Clostridium Anal

− − Bifidobacterium Anal

− − Streptococcus Skin and mouth

− − Staphylococcus Skin and mouth

− − Bacteroidales S24-7 Skin

− Stenotrophomonas Skin

− Gemellaceae Mouth

− Streptococcus Anal − −

Veillonella Anal − −

These results demonstrate that babies born vaginally showed a highlyvariable microbiota in all body sites (high inter-individual Unifracdistances), while C-section babies showed a microbiota with lowervariability, similar to that in maternal skin. C-section babies exposedto maternal inoculum showed communities resembling more those of thematernal vagina, with intermediate variability in relation to the othertwo groups. By day 30, oral and skin (but not fecal) microbiota clusterswith the corresponding maternal site. Therefore, exposing babiesdelivered by C-section to the vaginal microbiota of their motherspartially restores normal microbial colonization patterns to resemblevaginally delivered infants.

The invention thus provides that newborns exposed to the vaginal canalor gauze, acquire their mother's vaginal bacterial populations. Bloomsof bacteria that occur in newborns are still associated with maternalbacteria and with feeding mode. Babies are born with a bacterialdiversity that is higher than mother's vagina, and decreases sharplyafter birth, presumably due to the selective pressure of milk. Babiesthat are breastfed have oral and skin (forehead and arm) bacterialcommunities closer to their mother's (aureole). Feeding mode (breastfedor formula fed) can be predicted based on the closeness of baby (oral,forehead skin) and mother's (aureole) bacterial communities. Formulaallows higher colonization of several bacteria such as Leptotrichia.

It is demonstrated herein that disruption of the natural birth orfeeding process alters significantly the microbiota of babies, in acrucial developmental stage. Babies born by C-section can normalizetheir microbiota (e.g., resemble vaginally delivered babies) at birth,by being exposed to their mother's vaginal inoculum. The newbornbacterial diversity of the mouth and anal microbiota decreases soonafter birth, and is maintained remarkably low during the first month oflife. Breast feeding maintains closer baby-mother microbiota.

Example 3: Determine the Bacteria that Normalizes the Immune andMetabolic Development in Mice

The goal of the study is to determine if microbiota of C-section borninfants leads to higher inflammatory response and is more obesogenic.

Transfer of whole natural microbiota or mixed or pure isolates (i) fromhuman maternal vagina or (ii) from meconium/feces from babies bornvaginally are used to determine immune profile and metabolic responsesin GF mice, and are compared to GF mice who received a transfer ofmicrobiota (iii) from meconium/feces from babies delivered by C-section.

The aim is to determine the microbial taxa responsible for the observeddifferences, and to optimize for restoring healthy phenotypes, i.e.,minimizing the differences in responses of C-section related exposures,in relation to the vaginal control group. The final desired outcome isthe alleviation of exposures that increase the risks of C-sectionassociated disorders, using microbial exposures natural to mammals.

Example 4: Sequences

Sequences were obtained for region V4 of the 16S rRNA gene and comparedto the bacterial database “Greenhenes”. Taxa were identified with anidentity of >97%. OTUs grouped sequences sharing more than 97% identity.The data is provided in Table 6.

TABLE 6 Time Test- VD_1W_ CS_1W_ Representative OTU (weeks) SiteStatistic P Group mean mean Taxonomy sequence SEQ ID NO 4393532 1 Baby3.75 0. VD  1.4  0 k_Bacteria; TACGTAGGGAGC 1 anal 052807511 p_GAGCGTTATCCG Actinobacteria; GATTCATTGGGC c_Coriobacteriia; GTAAAGAGCGCo_C GTAGGCGGCCTC oriobacteriales; TCAAGCGGGATC f_Cor TCTAATCCGAGGiobacteriaceae; GCTCAACCCCCG g_Eggerthella; GCCGGATCCCGA s_lentaACTGGGAGGCTC GAGTTCGGTAGA GGCAGGCGGAA TTCCCGGTGTAG CGGTGGAATGCGCAGATATCGGGA AGAACACCGAT GGCGAAGGCAG CCTGCTGGGCCG CAACTGACGCTGAGGCGCGAAAG CTAGGGGAGCG AACAGG 947112 1 Baby 3. 0. VD 10  0 k_Bacteria;TACGTAGGGTGC 2 anal 71559633 053906366 p_ GAGCGTTGTCCG Actinobacteria;GAATTACTGGGC _Actinobacteria; GTAAAGAGCTCG o_A TAGGTGGTTTGTctinomycetales; CGCGTCGTCTGT f_Coryne- GAAATTCCGGGG bacteriaceae;CTTAACTCCGGG g_ CGTGCAGGCGAT Cotynebacterium; ACGGGCATAACT s_TGAGTACTGTAG GGGAGACTGGA ATTCCTGGTGTA GCGGTGAAATGC GCAGATATCAGGAGGAACACCGG TGGCGAAGGCG GGTCTCTGGGCA GTAACTGACGCT GAGGAGCGAAAGCATGGGGAGC GAACAGG 4468234 1 Baby 3. 0. VD 17.4  0 k_Bacteria;TACGGAGGATCC 3 anal 71559633 053906366 p_ GAGCGTTATCCG Bacteroidetes;GATTTATTGGGT c_Bacteroidia; TTAAAGGGAGC o_Bacteroidales; GTAGATGGATGT f_TTAAGTCAGTTG Bacteroidaceae; TGAAAGTTTGCG g_ GCTCAACCGTAA Bacteroides;AATTGCAGTTGA s_ TACTGGATGTCT TGAGTGCAGTTG AGGCAGGCGGA ATTCGTGGTGTAGCGGTGAAATGC TTAGATATCACG AAGAACTCCGAT TGCGAAGGCAG CCTGCTAGGCTGCAACTGACATTG AGGCTCGAAAGT GTGGGTATCAAA CAGG 4376828 1 Baby 3. 0. VD  1.2 0 k_Bacteria; TACGTAGGGCGC 4 anal 71559633 053906366 p_ AAGCGTTATCCGActinobacteria; GATTTATTGGGC c_ GTAAAGGGCTCG Actinobacteria;TAGGCGGCTCGT o_ CGCGTCCGGTGT Bifidobacteriales; GAAAGTCCATCG f_CTTAACGGTGGA Bifidobacteriaceae; TCTGCGCCGGGT g_Bifidobacterium;ACGGGCGGGCTT s_ GAGTGCGGTAG GGGAGACTGGA ATTCCCGGTGTA ACGGTGGAATGTGTAGATATCGGG AAGAACACCAA TGGCGAAGGCA GGTCTCTGGGCC GTTACTGACGCTGAGGAGCGAAA GCGTGGGGAGC GAACAGG 578016 1 Baby 3. 0. VD 14.2  0k_Bacteria; TACGGAGGATCC 5 anal 71559633 053906366 p_Bacteroidetes;GAGCGTTATCCG c_Bacteroidia; GATTTATTGGGT o_ TTAAAGGGTGCG Bacteroidales;TAGGCGGCCTTT f_ TAAGTCAGCGGT Porphyro- GAAAGTCTGTGG monadaceae;CTCAACCATAGA g_ ATTGCCGTTGAA Parabacteroides; ACTGGGGGGCTT s_distasonisGAGTATGTTTGA GGCAGGCGGAA TGCGTGGTGTAG CGGTGAAATGCT TAGATATCACGCAGAACCCCGATT GCGAAGGCAGC CTGCCAAGCCAT GACTGACGCTGA TGCACGAAAGCGTGGGGATCAA ACAGG 495067 1 Baby 3. 0. VD 19.4  0 k_Bacteria;TACGTAGGGTGC 6 anal 37155963 053906366 p_Actinobacteria; GAGCGTTGTCCGc_Actinobacteria; GAATTACTGGGC o_ GTAAAGAGCTCG Actinomycetales;TAGGCGGTTTGT f_ CACGTCGTCTGT Coryne- GAAATCCTAGGG bacteriaceae;CTTAACCCTGGA g_ CGTGCAGGCGAT Cotynebacterium; ACGGGCTGACTT s_GAGTACTACAGG GGAGACTGGAA TTTCTGGTGTAG CGGTGGAATGCA CAGATATCAGGAAGAACACCGAT GGCGAAGGCAG GTCTCTGGGTAG TAACTGACGCTG AGGAGCGAAAGCATGGGTAGCGA ACAGG 114510 1 Baby 3. 0. VD  4.8  0 k_Bacteria;TACGGAGGGTGC 7 anal 71559633 053906366 p_Proteobacteria; AAGCGTTAATCGc_Gammapro- GAATTACTGGGC teobacteria; GTAAAGCGCAC o_ GCAGGCGGTTTGEnterobacteriales: TTAAGTCAGATG f_ TGAAATCCCCGG Enterobacteriaceae;GCTCAACCTGGG g_; s_ AACTGCATCTGA TACTGGCAAGCT TGAGTCTCGTAG AGGGGGGTAGAATTCCAGGTGTA GCGGTGAAATGC GTAGAGATCTGG AGGAATACCGGT GGCGAAGGCGG 526804 4Baby 4.4 0. VD  1.6  0 k_Bacteria; TACGTAGGTCCC 8 anal 035938931p_Firmicutes; GAGCGTTATCCG c_Bacilli; GATTTATTGGGC o_ GTAAAGCGAGCLactobacillales; GCAGGCGGTTAG f_ ATAAGTCTGAAG Streptococcaceae;TTAAAGGCTGTG g_Streptococcus; GCTTAACCATAG s_ TACGCTTTGGAA ACTGTTTAACTTGAGTGCAGAAG GGGAGAGTGGA ATTCCATGTGTA GCGGTGAAATGC GTAGATATATGGAGGAACACCGG TGGCGAAAGCG GCTCTCTGGTCT GTAACTGACGCT GAGGCTCGAAAGCGTGGGGAGC GAACAGG 1082607 4 Baby 4. 0. VD  2.4  0 k_Bacteria;TACGTAGGGTGC 9 anal 367647059 036627535 p_Actinobacteria; GAGCGTTGTCCGc_ GAATTACTGGGC Actinobacteria; GTAAAGAGCTCG o_ TAGGCGGTTTGTActinomycetales; CACGTCGTCTGT f_Coryne- GAAATCCTAGGG bacteriaceae;CTTAACCCTGGA g_ CGTGCAGGCGAT Cotynebacterium; ACGGGCTGACTT s_GAGTACTACAGG GGAGACTGGAA TTTCTGGTGTAG CGGTGGAATGCA CAGATATCAGGAAGAACACCGAT GGCGAAGGCAG GTCTCTGGGTAG TAACTGACGCTG AGGAGCGAAAGCATGGGGAGCG AACAGG 495067 4 Baby 4. 0. VD 22  0 k_Bacteria; TACGTAGGGTGC10 anal 367647059 036627535 p_Actinobacteria; GAGCGTTGTCCG c_GAATTACTGGGC Actinobacteria; GTAAAGAGCTCG o_ TAGGCGGTTTGTActinomycetales; CACGTCGTCTGT f_Coryne- GAAATCCTAGGG bacteriaceae;CTTAACCCTGGA g_ CGTGCAGGCGAT Cotynebacterium; ACGGGCTGACTT s_GAGTACTACAGG GGAGACTGGAA TTTCTGGTGTAG CGGTGGAATGCA CAGATATCAGGAAGAACACCGAT GGCGAAGGCAG GTCTCTGGGTAG TAACTGACGCTG AGGAGCGAAAGCATGGGTAGCGA ACAGG 4425214 4 Baby 3. 0. VD 32.6 11 k_Bacteria;TACGTAGGTCCC 11 anal 348554033 067264075 p_Firmicutes; GAGCGTTGTCCGc_Bacilli; GATTTATTGGGC o_ GTAAAGCGAGC Lactobacillales; GCAGGCGGTTTGf_Strep- ATAAGTCTGAAG tococcaceae; TTAAAGGCTGTG g_Streptococcus;GCTCAACCATAG s_ TTCGCTTTGGAA ACTGTCAAACTT GAGTGCAGAAG GGGAGAGTGGAATTCCATGTGTA GCGGTGAAATGC GTAGATATATGG AGGAACACCGG TGGCGAAAGCGGCTCTCTGGTCT GTAACTGACGCT GAGGCTCGAAA GCGTGGGGAGC GAACAGG 4429335 4 Baby2. 0. VD  8.2 0. k_Bacteria; TACGTAGGGGGC 12 anal 879528986 08971226833333333 p_Firmicutes; TAGCGTTGTCCG c_Clostridia; GAATCACTGGGC o_GTAAAGGGTTCG Clostridiales; CAGGCGGAAAT f_ GCAAGTCAGGTG[Tissierellaceae]; TAAAAGGCAGT g_Peptoniphilus; AGCTTAACTACT s_GTAAGCATTTGA AACTGCATATCT TGAGAAGAGTA GAGGTAAGTGG AATTTTTAGTGTAGCGGTGAAAT GCGTAGATATTA AAAAGAATACC GGTGGCGAAGG CGACTTACTGGGCTCATTCTGACG CTGAGGAACGA AAGCGTGGGTA GCAAACAGG

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1. A method for restoring normal microbiota in an infant delivered byCesarean section, said method comprising administering to said infant atthe time of birth and/or within the first 4 months of life an effectiveamount of a vaginal microbiota inoculum, wherein said inoculum isobtained from the subject's mother or from a donor during the thirdtrimester of pregnancy before or at the time of giving birth.

2. The method of item 1, wherein the vaginal microbiota inoculum isadministered to the infant within the first 24 hours of life.

3. A method for restoring normal microbiota in a pre-term infant, saidmethod comprising administering to said infant at the time of birthand/or within the first 4 months of life an effective amount of avaginal microbiota inoculum, wherein said inoculum is obtained from thesubject's mother or from a donor during the third trimester of pregnancybefore or at the time of giving birth.

4. The method of item 3, wherein the vaginal microbiota inoculum isadministered to the newborn subject within the first hour of life.

5. A method for treating a disease in a subject associated with thesubject's delivery by Cesarean section or with the subject's pre-termbirth, said method comprising administering to said subject at the timeof birth and/or within the first 4 months of life a therapeuticallyeffective amount of a vaginal microbiota inoculum, wherein said inoculumis obtained from the subject's mother or from a donor during the thirdtrimester of pregnancy before or at the time of giving birth.

6. The method of item 5, wherein said disease is an inflammatory or anautoimmune disorder.

7. The method of item 5, wherein said disease is selected from the groupconsisting of autoimmune diseases, allergic diseases, infectiousdiseases, and rejection in organ transplantations.

8. The method of item 5, wherein said disease is selected from the groupconsisting of asthma, allergy, celiac disease, type 1 diabetes, obesity,necrotizing enterocolitis, inflammatory bowel disease (IBD), ulcerativecolitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoidarthritis, multiple sclerosis, graft vs. host disease following bonemarrow transplantation, osteoarthritis, juvenile chronic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, insulin dependent diabetes mellitus,thyroiditis, asthma, psoriasis, dermatitis scleroderma, atopicdermatitis, graft versus host disease, acute or chronic immune diseaseassociated with organ transplantation, sarcoidosis, and atherosclerosis.

9. The method of any one of items 1-8, wherein the vaginal microbiotainoculum is delivered to the mouth, nose, and/or skin of the infant.

10. The method of any one of items 1-8, wherein the vaginal microbiotainoculum is administered to the infant by a route selected from thegroup consisting of oral, topical, rectal, mucosal, sublingual, nasal,and via naso/oro-gastric gavage.

11. The method of any one of items 1-10, wherein the vaginal microbiotainoculum is administered to the infant by placing it on the maternalbreast and/or chest.

12. The method of any one of items 1-11, wherein the vaginal microbiotainoculum is delivered to the infant in a form of a liquid, foam, cream,spray, powder, or gel.

13. The method of any one of items 1-11, wherein the vaginal microbiotainoculum is delivered to the infant in a form of a composition whichcomprises (i) a carrier and/or excipient and/or (ii) one or moreprebiotic agents which stimulate growth and/or activity of one or morebacteria present in the composition.

14. The method of item 13, wherein the composition comprises a bufferingagent to adjust pH to the natural vaginal pH at the time of labor or toa pH of 3.5 to 7.

15. The method of item 13 or 14, wherein the composition comprises anexcipient or a carrier that optimizes the seeding of the transferredmicrobiota.

16. The method of any one of items 1-15, wherein the vaginal microbiotainoculum is obtained and/or delivered using an absorbent material ordevice.

17. The method of item 16, wherein the absorbent material or device isselected from the group consisting of gauze, sponge, and tampon.

18. The method of item 16 or 17, wherein the vaginal microbiota inoculumis transferred to said absorbent material or device by introducing saidabsorbent material or device in vagina prior to the birth or at the timeof Cesarean section.

19. The method of item 18, wherein said absorbent material or device isintroduced in the vagina for at least 5 minutes.

20. The method of any one of items 1-19, wherein said vaginal microbiotainoculum, after it is obtained from the subject's mother or the donor,is stored in a frozen form.

21. The method of any one of items 1-20, wherein said vaginal microbiotainoculum, after it is obtained from the subject's mother or the donor,is processed to isolate desired bacteria as single or mixed cultures andsuch mixed or single cultures are then administered to the infant.

22. The method of any one of items 1-21, wherein said vaginal microbiotainoculum is lyophilized after it is obtained from the subject's motheror the donor and reconstituted prior to the administration to theinfant.

23. The method of any one of items 1-12, wherein prior to obtainingvaginal microbiota inoculum from the newborn's mother or the donor, itis verified that said mother or donor does not have Group BStreptococcus (GB S), human immunodeficiency virus (HIV), and/orChlamydia.

24. The method of any one of items 1-23, wherein prior to obtainingvaginal microbiota inoculum from the newborn's mother or the donor, itis verified that said mother or donor does not have sexually transmitteddiseases.

25. The method of any one of items 1-24, wherein prior to obtainingvaginal microbiota from the newborn's mother or the donor, it isverified that said mother's or donor's vaginal pH is less than 4.5.

26. The method of any one of items 1-25, wherein the newborn's mother orthe donor has not been administered antibiotic compounds at least onemonth prior to the collection of the microbiota, has body mass index(BMI) between 18.5 and 24.9, and does not have irritable bowel disease,Crohn's disease, ulcerative colitis, irritable bowel syndrome, celiacdisease, colorectal cancer, and a family history of these diseases.

27. The method of any one of items 1-26, further comprising monitoringthe infant's microbiota after the administration of the vaginalmicrobiota inoculum by:

(a) determining a relative abundance of one or more bacterial taxa in amicrobiota sample obtained from the infant, and

(b) comparing the relative abundance(s) determined in step (a) to (i) apredetermined standard value or (ii) to the abundance(s) of the sametaxa in a control subject or (iii) to the average value of abundances ofthe same taxa in several control subjects.

28. The method of item 27, wherein the infant's microbiota sample isisolated from feces, skin, oral mucosa, conjunctive mucosa, or nasalmucosa.

29. The method of item 27 or item 28, wherein the control subject is avaginally delivered full-term healthy infant.

30. The method of item 29, wherein the control subject is born to amother who has not been administered antibiotic compounds at least onemonth prior to giving birth, has body mass index (BMI) between 18.5 and24.9, and does not have irritable bowel disease, Crohn's disease,ulcerative colitis, irritable bowel syndrome, celiac disease, colorectalcancer, and a family history of these diseases.

31. The method of any one of items 27-30, wherein determining therelative abundance of the bacterial taxa comprises a method selectedfrom the group consisting of quantitative polymerase chain reaction(qPCR), sequencing of bacterial 16S rRNA, shotgun metagenome sequencing,and metabolomics.

32. The method of any one of items 27-31, wherein the bacterial taxacomprise one or more taxa selected from the group consisting ofLactobacillus, Bacteriodales, Bacteroides, Parabacteroides,Bacteroidacea, Porphyromonadaceae, Coriobacteriales, Bifidobacterium,Clostridiaceae, Stenotrophomonas, and Gemella.

33. The method of item 30, wherein Bacteriodales is S24-7.

34. The method of any one of items 27-31, wherein the bacterial taxacomprise one or more taxa recited in Table 1A.

35. The method of any one of items 27-31, wherein the bacterial taxacomprise one or more species recited in Table 1B.

36. The method of any one of items 27-31, wherein the bacterial taxacomprise family Neisseriaceae.

37. A method for restoring normal microbiota in an infant delivered byCesarean section, said method comprising administering to said infant atthe time of birth and/or within the first 4 months of life an effectiveamount of a probiotic composition, wherein said probiotic composition(i) stimulates growth and/or activity of bacteria which areunder-represented in microbiota of said infant as compared to vaginallydelivered full-term infants, and/or (ii) inhibits growth and/or activityof bacteria which are over-represented in microbiota of said infant ascompared to vaginally delivered full-term infants.

38. A method for restoring normal microbiota in a pre-term infant, saidmethod comprising administering to said infant at the time of birthand/or within the first 4 months of life an effective amount of aprobiotic composition, wherein said probiotic composition (i) stimulatesgrowth and/or activity of bacteria which are under-represented inmicrobiota of said infant as compared to vaginally delivered full-terminfants, and/or (ii) inhibits growth and/or activity of bacteria whichare over-represented in microbiota of said infant as compared tovaginally delivered full-term infants.

39. A method for treating a disease in a subject associated with thesubject's delivery by Cesarean section or with the subject's pre-termbirth, said method comprising administering to said subject at the timeof birth and/or within the first 4 months of life a therapeuticallyeffective amount of a probiotic composition, wherein said probioticcomposition (i) stimulates growth and/or activity of bacteria which areunder-represented in microbiota of said infant as compared to vaginallydelivered full-term infants, and/or (ii) inhibits growth and/or activityof bacteria which are over-represented in microbiota of said infant ascompared to vaginally delivered full-term infants.

40. The method of item 39, wherein said disease is an inflammatory or anautoimmune disorder.

41. The method of item 39, wherein said disease is selected from thegroup consisting of autoimmune diseases, allergic diseases, infectiousdiseases, and rejection in organ transplantations.

42. The method of item 39, wherein said disease is selected from thegroup consisting of asthma, allergy, celiac disease, type 1 diabetes,obesity, necrotizing enterocolitis, inflammatory bowel disease (IBD),ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis,rheumatoid arthritis, multiple sclerosis, graft vs. host diseasefollowing bone marrow transplantation, osteoarthritis, juvenile chronicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, insulin dependentdiabetes mellitus, thyroiditis, asthma, psoriasis, dermatitisscleroderma, atopic dermatitis, graft versus host disease, acute orchronic immune disease associated with organ transplantation,sarcoidosis, and atherosclerosis.

43. The method of any one of items 37-42, wherein said probioticcomposition comprises one or more bacterial strains from one or moretaxa selected from the group consisting of Lactobacillus, Bacteriodales,Bacteroides, Parabacteroides, Bacteroidacea, Porphyromonadaceae,Coriobacteriales, Bifidobacterium, Clostridiaceae, Stenotrophomonas, andGemella.

44. The method of item 43, wherein Bacteriodales is S24-7.

45. The method of any one of items 37-42, wherein said probioticcomposition comprises one or more bacterial strains from one or moretaxa recited in Table 1A.

46. The method of any one of items 37-42, wherein said probioticcomposition comprises one or more bacterial strains recited in Table 1B.

47. The method of any one of items 37-42, wherein said probioticcomposition comprises one or more bacterial strains from the familyNeisseriaceae.

48. The method of any one of items 37-47, wherein said probioticcomposition comprises one or more bacterial strains which can be foundin a healthy vaginal microbiota from a pregnant woman in the thirdtrimester of pregnancy before or at the time of giving birth.

49. The method of item 48, wherein the woman has not been administeredantibiotic compounds within at least one month prior to isolation ofbacteria, has body mass index (BMI) between 18.5 and 24.9, does not haveGroup B Streptococcus (GBS), human immunodeficiency virus (HIV),Chlamydia, and/or sexually transmitted diseases, has vaginal pH lessthan 4.5, and does not have irritable bowel disease, Crohn's disease,ulcerative colitis, irritable bowel syndrome, celiac disease, colorectalcancer and a family history of these diseases.

50. The method of any one of items 37-49, wherein said probioticcomposition comprises live bacterial cells.

51. The method of any one of items 37-49, wherein said probioticcomposition comprises one or more components selected from the groupconsisting of conditionally lethal bacterial cells, inactivatedbacterial cells, killed bacterial cells, spores, recombinant carrierstrains, cell extract, and bacterially-derived products.

52. The method of item 51, wherein the bacterially-derived product is abacterial antigen or a bacterial metabolic product.

53. The method of any one of items 37-52, wherein said probioticcomposition comprises (i) a carrier and/or excipient and/or (ii) one ormore prebiotic agents which stimulate growth and/or activity of one ormore bacteria present in the composition.

54. The method of any one of items 37-53, wherein said probioticcomposition is reconstituted from a lyophilized preparation.

55. The method of any one of items 37-54, wherein the probioticcomposition comprises a buffering agent to adjust pH to the naturalvaginal pH at the time of labor or to a pH of 3.5 to 7.

56. The method of item 53, wherein the probiotic composition comprisesan excipient or a carrier that optimizes the seeding of one or morebacterial strains contained in said probiotic composition.

57. The method of any one of items 37-56, wherein the probioticcomposition is delivered to the mouth, nose, and/or skin of the infantand/or by placing it on the maternal breast and/or chest.

58. The method of any one of items 37-56, wherein the probioticcomposition is administered to the infant by a route selected from thegroup consisting of oral, topical, rectal, mucosal, sublingual, nasal,and via naso/oro-gastric gavage.

59. The method of any one of items 37-58, wherein the probioticcomposition is delivered to the infant in a form of a liquid, foam,cream, spray, powder, or gel.

60. The method of any one of items 37-59, wherein the probioticcomposition is delivered using an absorbent material or device.

61. The method of item 60, wherein the absorbent material or device isselected from the group consisting of gauze, sponge, and tampon.

62. The method of any one of items 37-60, wherein the probioticcomposition comprises a buffering agent.

63. The method of item 62, wherein the buffering agent comprises sodiumbicarbonate, infant formula or sterilized human milk.

64. The method of any one of items 37-63, wherein the probioticcomposition is administered conjointly with a prebiotic which stimulatesgrowth and/or activity of one or more bacteria contained in theprobiotic composition.

65. The method of item 64, wherein the prebiotic is selected from thegroup consisting of fructooligosaccharides (FOS),galactooligosaccharides (GO S), human milk oligosaccharides (HMO),Lacto-N-neotetraose, D-Tagatose, xylo-oligosaccharides (XOS),arabinoxylan-oligosaccharides (AXOS), N-acetylglucosamine,N-acetylgalactosamine, glucose, arabinose, maltose, lactose, sucrose,cellobiose, amino acids, alcohols, resistant starch (RS), and anymixtures thereof.

66. The method of item 64, wherein the prebiotic is derived frommicroorganisms that show stimulation by human milk components.

67. The method of any one of items 64-66, wherein the probiotic andprebiotic are administered in one composition, or simultaneously as twoseparate compositions, or sequentially.

68. A method for diagnosing an abnormal microbiota development in aninfant, comprising:

(a) determining a relative abundance of one or more bacterial taxa in amicrobiota sample obtained from the infant, and

(b) comparing the relative abundance(s) determined in step (a) to (i) apredetermined standard value or (ii) to the abundance(s) of the sametaxa in a control subject or (iii) to the median value of abundances ofthe same taxa in several control subjects, wherein the control subjectis a vaginally delivered full-term healthy infant.

69. The method of item 68, wherein determining the relative abundance ofthe bacterial taxa comprises a method selected from the group consistingof quantitative polymerase chain reaction (qPCR), sequencing ofbacterial 16S rRNA, shotgun metagenome sequencing, and metabolomics.

70. The method of item 68 or 69, wherein the bacterial taxa comprise oneor more taxa selected from the group consisting of Lactobacillus,Bacteriodales, Bacteroides, Parabacteroides, Bacteroidacea,Porphyromonadaceae, Coriobacteriales Bifidobacterium, Clostridiaceae,Stenotrophomonas, and Gemella.

71. The method of item 70, wherein Bacteriodales is S24-7.

72. The method of item 68 or 69, wherein the bacterial taxa comprise oneor more taxa present in a healthy vaginal microbiota from a pregnantwoman in the third trimester of pregnancy before or at the time ofgiving birth.

73. The method of item 68 or 69, wherein the bacterial taxa comprise oneor more taxa recited in Table 1A.

74. The method of item 68 or 69, wherein the bacterial taxa comprise oneor more species recited in Table 1B.

75. The method of item 68 or 69, wherein the bacterial taxa comprisefamily Neisseriaceae.

76. The method of any one of items 1-75, 103, and 104, wherein theinfant or subject is human.

77. A composition comprising (i) a vaginal microbiota inoculum and (ii)a carrier and/or excipient and/or one or more prebiotic agents whichstimulate growth and/or activity of one or more bacteria present in theinoculum.

78. A probiotic composition comprising (a) one or more bacterial strainsand (b) a carrier and/or excipient and/or one or more prebiotic agentswhich stimulate growth and/or activity of one or more of said bacterialstrains, wherein said probiotic composition (i) stimulates growth and/oractivity of bacteria which are under-represented in microbiota of aninfant delivered by by Cesarean section or born prematurely as comparedto vaginally delivered full-term healthy infants, and/or (ii) inhibitsgrowth and/or activity of bacteria which are over-represented inmicrobiota of said infant as compared to vaginally delivered full-termhealthy infants.

79. The composition of item 78, which comprises two or more differentbacterial strains.

80. The composition of any one of items 77-79, which comprises bacteriafrom one or more taxa selected from the group consisting ofLactobacillus, Bacteriodales, Bacteroides, Parabacteroides,Bacteroidacea, Porphyromonadaceae, Coriobacteriales, Bifidobacterium,Clostridiaceae, Stenotrophomonas, and Gemella.

81. The composition of item 80, wherein Bacteriodales is S24-7.

82. The composition of any one of items 77-79, which comprises bacteriafrom one or more taxa recited in Table 1A.

83. The composition of any one of items 77-79, which comprises bacteriafrom one or more species recited in Table 1B.

84. The composition of any one of items 77-79, which comprises bacteriafrom family Neisseriaceae.

85. The composition of any one of items 77-84, wherein the compositioncomprises a buffering agent to adjust pH to the natural vaginal pH atthe time of labor or to a pH of 3.5 to 7.

86. The composition of any one of items 77-85, wherein the compositioncomprises an excipient or a carrier that optimizes the seeding of one ormore bacterial strains contained in the composition.

87. The composition of any one of items 77-86, wherein said compositionis formulated for storage in a frozen form.

88. The composition of any one of items 77-87, wherein said compositionis a lyophilized composition.

89. The composition of any one of items 77-88, wherein said compositioncomprises one or more bacterial strains which can be found in a healthyvaginal microbiota from a pregnant woman in the third trimester ofpregnancy before or at the time of giving birth.

90. The composition of item 89, wherein the woman has not beenadministered antibiotic compounds at least one month prior to isolationof bacteria, has body mass index (BMI) between 18.5 and 24.9, does nothave Group B Streptococcus (GBS), human immunodeficiency virus (HIV),Chlamydia, and/or sexually transmitted diseases, has vaginal pH lessthan 4.5, and does not have irritable bowel disease, Crohn's disease,ulcerative colitis, irritable bowel syndrome, celiac disease, colorectalcancer or a family history of these diseases.

91. The composition of any one of items 77-90, wherein said compositioncomprises live bacterial cells.

92. The composition of any one of items 77-90, wherein said compositioncomprises one or more components selected from the group consisting ofconditionally lethal bacterial cells, inactivated bacterial cells,killed bacterial cells, spores, recombinant carrier strains, cellextract, and bacterially-derived products.

93. The composition of item 92, wherein the bacterially-derived productis a bacterial antigen or a bacterial metabolic product.

94. The composition of any one of items 77-93, wherein said compositionis formulated for delivery to the mouth, nose, and/or skin of the infantand/or for placing it on the maternal breast and/or chest.

95. The composition of any one of items 77-93, wherein said compositionis formulated for delivery by a route selected from the group consistingof oral, topical, rectal, mucosal, sublingual, nasal, and vianaso/oro-gastric gavage.

96. The composition of any one of items 77-95, wherein the compositionis in a form of a liquid, foam, cream, spray, powder, or gel.

97. The composition of any one of items 77-96, wherein the compositioncomprises a buffering agent.

98. The composition of item 97, wherein the buffering agent comprisessodium bicarbonate, infant formula or sterilized human milk.

99. The composition of any one of items 77-98, wherein the compositioncomprises a prebiotic which stimulates growth and/or activity of one ormore bacteria contained in the composition.

100. The method of item 99, wherein the prebiotic is selected from thegroup consisting of fructooligosaccharides (FOS),galactooligosaccharides (GO S), human milk oligosaccharides (HMO),Lacto-N-neotetraose, D-Tagatose, xylo-oligosaccharides (XOS),arabinoxylan-oligosaccharides (AXOS), N-acetylglucosamine,N-acetylgalactosamine, glucose, arabinose, maltose, lactose, sucrose,cellobiose, amino acids, alcohols, resistant starch (RS), and anymixtures thereof.

101. An absorbent material or device comprising the composition of anyone of items 77-100.

102. The material or device of item 101, wherein the material or deviceis selected from the group consisting of gauze, sponge, and tampon.

103. A method for restoring normal microbiota in an infant delivered byCesarean section, said method comprising administering to said infant atthe time of birth and/or within the first 4 months of life an effectiveamount of the composition of any one of items 77-100.

104. A method for restoring normal microbiota in a pre-term infant, saidmethod comprising administering to said infant at the time of birthand/or within the first 4 months of life an effective amount of thecomposition of any one of items 77-100.

105. A method for treating a disease in a subject associated with thesubject's delivery by Cesarean section or with the subject's pre-termbirth, said method comprising administering to said subject at the timeof birth and/or within the first 4 months of life a therapeuticallyeffective amount of the composition of any one of items 77-100.

106. The method of item 105, wherein the subject is human.

107. The method of any one of items 1-76, 103 and 104, wherein theinfant is a newborn.

108. A device to collect maternal vaginal microbiota from the vaginalcanal of a patient, comprising:

a housing forming a cavity;

an absorbent material disposed within the cavity; and

a deployment element disposed in the housing and movable along a lengthof the housing;

wherein the movement of the deployment element displaces the absorbentmaterial out of the cavity and into the vaginal canal.

109. The device of item 108, wherein the absorbent material comprises amaterial that cannot be used as at least one of a menstrual pad, tampon,or urine pad.

110. The device of any one of items 108-109, wherein the housing and thedeployment elements are sized to deliver the absorbent material withinthe lower ⅓ of the vaginal canal.

111. The device of any one of items 108-110, wherein the absorbentmaterial lacks a retrieval interface.

112. The device of any one of items 108-110, wherein the absorbentmaterial comprises a retrieval interface.

113. The device of item 112, wherein the retrieval interface is atether, monofilament plastic line, or reattachable interface.

114. The device of any one of items 112-113, wherein the retrievalinterface has a length and wherein at a full length the retrievalinterface does not exit the vaginal canal.

115. The device of any one of items 112-113, wherein the retrievalinterface has a length and wherein at a full length the retrievalinterface does exit the vaginal canal.

116. The device of any one of items 108-115, further comprising aretrieval element.

117. The device of any one of item 116, wherein the retrieval elementcaptures a deployed absorbent material and removes the deliveredabsorbent material from the vaginal canal.

118. The device of any one of items 108-117, wherein the deploymentelement acts as the retrieval element.

119. The device of any one of items 108-110 and 112-118, wherein theabsorbent material comprises a retrieval interface and wherein theretrieval element captures the deployed absorbent material byinteracting with the retrieval interface.

120. The device of any one of items 116-119, wherein the retrievalelement is disposed within the housing and captures the deployedabsorbent material and returns the absorbent material to the cavity forthe removal of the absorbent material from the vaginal canal.

121. The device of any one of items 108-120, further comprising a secondhousing forming a second cavity;

wherein a retrieval element is disposed within the second housing andcaptures the deployed absorbent material and returns the absorbentmaterial to the second cavity for the removal of the absorbent materialfrom the vaginal canal.

122. The device of any one of items 108-121, wherein at least one of thehousing, the cavity, the absorbent material and the deployment elementare sterile.

123. The device of any one of items 108-122, wherein the housing furthercomprises a seal around at least one of the cavity and the absorbentmaterial to maintain sterility.

124. The device of item 123, wherein the deployment element breaks theseal when deploying the absorbent material.

125. A method for collecting vaginal microbiota using the device of anyone of items 108-124.

126. The method of item 125, wherein the vaginal microbiota is stored.

127. The method of item 125 or 126, wherein the vaginal microbiota isprocessed to isolate desired bacteria.

128. The method of item 126, wherein the vaginal microbiota is processedto isolate desired bacteria after being stored.

129. The method of item 126, wherein the vaginal microbiota is processedto isolate desired bacteria prior to being stored.

130. A method for collecting and administering vaginal microbiota usingthe device of any one of items 108-124.

131. The method of item 130, wherein the vaginal microbiota is stored.

132. The method of item 130 or 131, wherein the vaginal microbiota isprocessed to isolate desired bacteria.

133. The method of item 131, wherein the vaginal microbiota is processedto isolate desired bacteria after being stored.

134. The method of item 131, wherein the vaginal microbiota is processedto isolate desired bacteria prior to being stored.

135. The method of any one of items 130-134, wherein the vaginalmicrobiota is administered to an infant delivered by Cesarean section orborn prematurely.

136. The method of any one of items 130-135, wherein the vaginalmicrobiota is administered at the time of birth and/or within the first4 months of life.

137. The method of any one of items 125-136, wherein the vaginalmicrobiota is obtained from the subject's mother or from a donor duringthe third trimester of pregnancy before or at the time of giving birth.

138. The method of any one of items 130-137, wherein a therapeuticallyeffective amount of vaginal microbiota is administered to treat adisease associated with an inflammatory or an autoimmune disorder.

139. The method of any one of items 130-138, wherein a therapeuticallyeffective amount of vaginal microbiota is administered to restore normalmicrobiota colonization patterns in an infant.

While the foregoing has described what are considered to be the bestmode and/or other examples, it is understood that various modificationsmay be made therein and that the subject matter disclosed herein may beimplemented in various forms and examples, and that the teachings may beapplied in numerous applications, only some of which have been describedherein. It is intended by the following claims to claim any and allapplications, modifications and variations that fall within the truescope of the present teachings.

What is claimed is:
 1. A device to collect vaginal microbiota from thevaginal canal of a patient, comprising: a housing forming a cavity; anabsorbent material disposed within the cavity; a deployment elementdisposed in the housing and movable along a length of the housing,wherein the movement of the deployment element removably displaces theabsorbent material out of the cavity and into the vaginal canal; and aretrieval element separate from the deployment element and configured tocapture a deployed absorbent material and remove the deployed absorbentmaterial from the vaginal canal, wherein the deployed absorbent materialis configured such that a proximal end of the deployed absorbentmaterial does not extend beyond a distal end of the retrieval elementduring removal of the deployed absorbent material.
 2. The device ofclaim 1, wherein the housing and the deployment element are sized toremovably deliver the absorbent material within the lower ⅓ of thevaginal canal.
 3. The device of claim 1, wherein the absorbent materiallacks a retrieval interface.
 4. The device of claim 1, wherein theabsorbent material comprises a retrieval interface having a length, andat a full length the retrieval interface does not exit the vaginalcanal.
 5. The device of claim 1, further comprising a reattachableinterface, wherein the retrieval element is configured to capture thedeployed absorbent material via the reattachable interface.
 6. Thedevice of claim 1, wherein the retrieval element is disposed within thehousing and is configured to capture the deployed absorbent material andreturn the absorbent material to the cavity for the removal of theabsorbent material from the vaginal canal.
 7. The device of claim 1,further comprising a second housing forming a second cavity; wherein theretrieval element is disposed within the second housing and isconfigured to capture the deployed absorbent material and return theabsorbent material to the second cavity for the removal of the absorbentmaterial from the vaginal canal.
 8. The device of claim 1, wherein atleast one of the housing, the cavity, the absorbent material and thedeployment element are sterile.
 9. The device of claim 8, wherein thehousing further comprises a seal around at least one of the cavity andthe absorbent material to maintain sterility.
 10. The device of claim 9,wherein the deployment element is configured to break the seal whendeploying the absorbent material.
 11. A method comprising: Collectingvaginal microbiota using the device of claim
 1. 12. The device of claim5, wherein: the reattachable interface comprises a first componentdisposed on the proximal end of the deployed absorbent material and asecond component disposed on the distal end of the retrieval element,and the first and second components are configured to engage duringremoval of the deployed absorbent material.
 13. The device of claim 12,wherein the first and second components comprise one or more magnets.14. The device of claim 12, wherein the first and second componentscomprise one or more hooks and/or loops.
 15. The device of claim 1,wherein the retrieval element is configured to remove the deployedabsorbent material from the vaginal canal via suction.